Functional Analysis of Barley MLA-triggered Disease ... · 2.2.4. Protein analysis 2.2.4.1....

Functional Analysis of Barley MLA-triggered

Disease Resistance to the Powdery Mildew Pathogen

Inaugural-Dissertation

zur

Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät

der Universität zu Köln

vorgelegt von

Qian-Hua Shen

aus Nanchang, China

Köln, May 2004

Berichterstatter: Prof. Dr. Paul Schulze-Lefert

Prof. Dr. Martin Hülskamp

Prüfungsvorsitzender: Prof. Dr. Reinhard Krämer

Tag der mündlichen Prüfung: 05. July 2004

Abbreviations

AD activation domain

Adh alcohol dehydrogenase

AGT appressorial germ tube

APP appressorium

ATP adenosine 5-triphosphate

Avr avirulence

BAC bacteria artificial chromosome

BD binding domain

Bgh Blumeria graminis f sp hordei

bp base pair

CA carbonic anhydrase

CC coiled-coil

CFU colony forming unit

CHORD cysteine- and histidine- rich domain

CS CHORD and SGT1 motif

CSN COP9 signalosome

CT carboxy-terminal non-LRR region

cv. cultivar

dATP deoxyadenosinetriphosphate

dCTP deoxycytidinetriphosphate

DEPC diethylpolycarbonate

dGTP deoxyguanosinetriphosphate

DMF dimethylformamide

DMSO dimythysulfoxide

DNA deoxyribonuleic acid

DNase deoxyribonuclease

dNTP deoxynucleosidetriphosphate

dsRNAi double-stranded RNA interference

DTT dithiothrietol

dTTP dioxythimydinetriphosphate

ECL enhanced chemiluminecence

E. coli Escherichia coli

EDTA ethylenediaminetetraacetic acid

EtBr ethidium bromide

EtOH ethanol

g gram

Gal galactose

GAL1 galactokinase

GUS β-glucuronidase

h hour

HR hypersensitive response

HRP horseradish peroxidase

HSP90 heat shock protein 90

IgG Immunoglobulin gamma chain

IT infection phenotype

kb kilobase (s)

kDa kilodalton (s)

LacZ β-galactosidase

L litre

LiAc lithium acetate

LRR leucine-rich repeat

min Minute(s)

Mla mildew-resistance locus A

mmol millimolar

mRNA messenger ribonucleic acid

NB nucleotide binding site

ng nanogram

ORF open reading frame

PAGE polyacrylamide gel electrophoresis

PCR polymerase chain reaction

PEG polyethylene glycol

pg picogram

PGT primary germ tube

pmol picomolar

Raf raffinose

Rar1 required for Mla12 resistance

RGH resistance gene homolog

RNA ribonucleic acid

RT room temperature

RT-PCR reverse transcription-polymerase chain reaction

SAR systemic acquired resistance

SCF SKP1/CULLIN/F-box protein

SD synthetic dropout (media)

SDS sodium dodecyl sulphate

SDS-PAGE SDS polyacrylamide gel electrophoresis

sec second(s)

Sgt1 suppressor of G-two allele of skp1

SKP1 suppressor of kinetochore protein

TIR Drosophila Toll and human interleukin-1 receptor

TPR tetratricopeptide repeat

TRIS Tris-(hydroxymethyl)-aminomethane

U unit

O/N over night

V Volt

vir virulence

VIGS virus induced gene silencing

%(v/v) volume-percent

%(w/v) weight-percent

WT wild type

X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

X-Gluc 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid

I

Table of Contents

1. General Introduction 1 1.1. Barley powdery mildew disease 1

1.1.1. The host plant

1.1.2. The pathogen

1.1.3. Fungal development and disease symptoms

1.2. Plant race-specific resistance genes 4

1.3. The Mla locus and Mla resistance genes 11

1.3.1. Molecular characterization of the Mla locus and Mla genes

1.3.2. Mla-mediated infection types

1.4. Rar1 and Sgt1--- two genes required for disease resistance 13

1.4.1. The Rar1 gene

1.4.2. The Sgt1 gene

1.5. Current models of pathogen recognition in plant disease resistance 17

1.5.1. Direct physical R protein-AVR effector interaction

1.5.2. The Guard Hypothesis

2. Material and Methods 20 2.1. Materials 20

2.1.1. Antibiotics 20

2.1.2. Antibodies 20

2.1.3. Bacterial strains 20

2.1.3.1. E. coli strains

2.1.4. Yeast strains 21

2.1.5. Fungal strains 21

2.1.6. Plant materials 22

Contents

II

2.1.7. Vectors 23

2.1.8. Oligonucleotides 24

2.1.9. Enzymes 26

2.1.9.1. Restriction enzymes

2.1.9.2. Nucleic acid modifying enzymes

2.1.10. Chemicals 26

2.1.11. Media 27

2.1.12. Buffers and solutions 28

2.1.13. General buffers and solutions

2.1.14. DNA buffers

2.1.15. Western buffers

2.2. Methods 33

2.2.1. Nucleic acid manipulations

2.2.1.1. Polymerase chain reaction (PCR) amplification

2.2.1.2. Restriction endonuclease digestion of DNA

2.2.2. DNA analysis 34

2.2.2.1. Plasmid DNA isolations

2.2.2.2. Plant genomic DNA isolation

2.2.2.3. Isolation of DNA fragment from Agrose-gel

2.2.2.4. DNA sequencing

2.2.2.5. DNA sequence analysis

2.2.2.6. Database searching

2.2.3. RNA analysis 36

2.2.3.1. Isolation of total RNA from plant tissues

2.2.3.2. RT-PCR

2.2.4. Protein analysis 38

2.2.4.1. Denaturing SDS-polyacrylamide gel electrophoresis

2.2.5. Transformation of E. coli 40

2.2.5.1. Preparation of electro-competent E. coli cells

2.2.5.2. Transformation of electro-competent E. coli cells

Contents

III

2.2.6. High-efficiency transformation of yeast competent cells 41

2.2.6.1 The protocol

2.2.7. Yeast two-hybrid screening via interaction mating methods 42

2.2.8. Single-cell transient assay in barley epidermal cells using

particle bombardment 43

3. Isolation and Characterization of a New Mla

Resistance Specificity: Mla12 46

3.1. Introduction 46

3.2. Characterization of susceptible mla12 mutant alleles 47

3.3. Over-expression of Mla12 alters the resistance kinetics

but retains Rar1 dependence 49

3.3.1. Over-expression of Mla12 alters the resistance kinetics

3.3.2. Over-expression of Mla12 complements mutant phenotypes

but retains Rar1 dependence

3.4. Sgt1 is required for Mla12 resistance 52

3.5. Context-dependent function of conserved MLA residues Leu631 and Lys916 53

3.6. Discussion: Altering resistance response kinetics by Mla dosage 55

4. Structure and Function Analysis of MLA Protein

by Domain Swapping: the LRR-CT Unit in MLA1

and MLA6 Determines Recognition Specificity 57


4.2. The CT domain in MLA proteins is also subject to diversifying

selection 57

4.3. Domain swaps between MLA1 and MLA6 reveal determinants

for recognition specificity reside in LRR-CT unit 59

4.4. Uncoupling the RAR1 requirement from MLA6 recognition

specificity 61

Contents

IV

4.5. SGT1 is associated with RAR1 in MLA mediated resistances 62

5. RAR1 is not Sufficient to Increase MLA Steady-state

Protein Levels in Saccharomyces cerevisiae 65


5.2. RAR1 does not alter MLA steady-state protein levels in yeast at

standard growth temperature 66

5.3. RAR1 does not impair MLA protein abundance in yeast at elevated

temperature 69

5.4. Discussion 69

6. Identification of MLA Interactors Using Yeast

Two-Hybrid Selection 73

6.1. The LexA yeast two-hybrid system and interaction mating method 73

6.1.1. The LexA yeast two-hybrid system

6.1.2. The interaction mating method

6.2. Construction of multiple LexA-MLA fusion baits using domains or full-

length sequences of MLA1 and MLA6 75

6.3. Transforming yeast strain EGY48 (MATα) with bait plasmid and

Characterization of bait strains 77

6.4. A barley prey library suitable for yeast two-hybrid selection by mating

type 79

6.5. Library screenings using interaction mating methods 79

6.6. Characterization of cDNA clones isolated from the prey library 80

6.6.1. Eliminating false positive clones

6.6.2. Discriminating non-redundant clones from redundant ones

6.6.3. MLA proteins/domains associate with structurally distinct host

proteins

6.7. Summary and perspective 88

7. General Discussion 93

Contents

V

7.1. Allelic variants encode MLA R proteins 93

7.2. Determinants of MLA recognition specificity 95

7.3. Potential roles of RAR1 and SGT1 in MLA-mediated resistances 96

7.3.1. The Ubiquitin/26S proteasome degradation pathway and MLA-

mediated resistance are connected to RAR1/SGT1

7.3.2. RAR1/SGT1 may act as co-chaperones in MLA-mediated

resistance

7.4. Direct versus indirect AVRMLA recognition 103

8. Summary 106

9. Zusammenfassung 108

10. Literature cited 111

11. Appendix: Publications 134

12. Acknowledgements 148

13. Lebenslauf 150

General introduction

1

1. General Introduction

1.1. Barley powdery mildew disease

1.1.1. The host plant

The host plant barley, Hordeum vulgare L. emend. Bowden, belongs to

the grass family Gramineae, tribe Hordeae (von Bothmer and Jacobsen,

1985). Barley is grown in many parts of the world, mostly in temperate

regions. It was suggested that the progenitors of barley originated from Asia in

the Israel-Jordan area; the world centre for genetic diversity is Ethiopia (von

Bothmer and Jacobsen, 1985; Badr et al., 2000; Salamini et al., 2002).

Cultivated barley has either a winter or a spring growth habit (winter and

spring barley, respectively). Barley is a diploid, self-pollinator with seven pairs

of chromosomes and has been extensively studied both genetically and

cytologically (Jogensen, 1994; Ramage, 1985). Like other crops, barley often

suffers from various diseases. One common fungal pathogen is the powdery

mildew disease whose spread is promoted by the relatively long vegetation

period and cool and humid climate in the northern hemisphere. It is estimated

that the powdery mildew disease can cause ~10% of yield losses in cooler

climates like in Europe in the absence of fungicides.

1.1.2. The pathogen

Powdery mildew disease in grass species is caused by Blumeria

graminis (= Erysiphe graminis). This fungus is strictly adapted into formae

speciales colonizing individual genera of the grass family, e.g. Blumeria

graminis f sp hordei (Bgh) is the causal agent of powdery mildew disease on

barley. Bgh successfully reproduces on wild and cultivated species of


2

Hordeum but fails to colonize other closely related cereals, such as wheat,

rye, or oats (Mathre,1982). The molecular basis of this narrow host range of

Bgh is not understood. The ascomycete Bgh is an obligate biotrophic

pathogen on barley, i.e. the fungus can reproduce only on living host tissue.

During its life cycle, the haploid form prevails except for a short diploid phase

after mating that includes the formation of cleistothecia and sexual

reproduction leading to ascospore formation. The common form of asexual

reproduction involves the formation of conidiophores that produce haploid

spores, called conidiospores. These spores are dispersed by wind and will

initiate a new infection cycle upon landing on a leaf blade or leaf sheath of

neighbouring plants. Airborne spores can migrate hundreds of kilometres

(Jørgensen, 1994; Thordal-Christensen et al., 2000).

Bgh attacks all aerial parts of the plant and infects only the epidermal

cell layer. Growth of fungal mycelium on the leaf surface leads to the powdery

appearance. The optimal temperature for development of Bgh is 20 oC.

1.1.3. Fungal development and disease symptoms

Under favourable conditions, the conidia will progress through a

germination phase including the formation of a primary and appressorial germ

tube (PGT and AGT, respectively). The AGT will swell at the end to form a

mature appressorium (APP). An appressorial infection peg is produced

beneath the APP to penetrate the plant cell wall and to make contact with the

plasma membrane of a leaf epidermal cell. Successful penetration leads to

the formation of a haustorium (a specialized feeding organ of Bgh) by

invagination of the plant plasma membrane. Subsequently elongating

secondary hyphae are formed on the leaf surface. Fungal growth at the leaf

surface typically results in the formation of a single powdery mildew colony

and eventually the formation of a new generation of conidia. The whole

asexual life cycle takes approximately 5-6 days (Boyd et al, 1995; Thordal-

Christensen et al., 2000). The Bgh haustorium is the sole fungal structure that


3

Con id iu m

PG T AG T

APP

ESH

~4 hp i

0 hp i

~11 hp i

~14 hp i

~24 hp i

~40 hp i

~72 hp i

P M

Con id iu m

Hau storium

. Figure 1. Diagram of the development of Blumeria graminis f sp hordei on barley leaf

epidermis. AGT, appressorial germ tube; APP, appressorium; ESH, elongating secondary hyphae; hpi,

hours post infection; PGT, primary germ tube; PM, plasma membrane; Modified from Body et

al,1995; Thordal-Christensien et al, 2000.


4

is in intimate physical contact with a host membrane (called extra-haustorial

membrane) and produces finger-like appendages when mature. The

haustorium complex (haustorium, extra-haustorial membrane, and extra-

hautorial matrix) is crucial for nutrient retrieval and mycelial growth on the leaf

surface (Fig.1; Schulze-Lefert and Vogel, 2000).

In interactions that lead to disease (frequently called compatible

interactions) or to immune responses (called incompatible interactions), it is

not unusual to observe at interactions sites on the same leaf different stages

of fungal development. However, the relative frequencies of the various

developmental stages differ and this often allows macroscopic discrimination

of five infection types (ITs), ranging from IT0, IT1, IT2 and IT3 (frequently

seen in incompatible interactions) to IT4 (compatible interaction) (Boyd et al,

1995; Thordal-Christensen et al., 2000). IT0 denotes an immune response

with no visible fungal growth and no immune response symptoms visible by

the naked eye. IT1 to IT3 denote infection types with increasing amounts of

fungal mycelium without sporulation and an increasing area of plant cells at

infection sites that die as part of the resistance response (‘necrotic flecks’).

IT4 corresponds to profuse colony growth including sporulation and lack of

recognizable immune responses at infection sites.

1.2. Plant race-specific resistance genes

Plants evolved different mechanisms to defend themselves against

microbial pathogens. A widespread form of plant immunity is race-specific

resistance that is governed by specific interactions involving gene pairs in

plant and pathogen (‘gene-for-gene’ interaction; Flor, 1971). In these cases, a

disease resistance response is triggered in the presence of matching

pathogen Avr (avirulence) and plant disease resistance (R) genes (Flor,

1971). At the species level, natural polymorphisms at R and Avr loci make it

possible to discriminate numerous plant lines and pathogen isolates,

respectively. A loss or alteration of either R or Avr gene leads to disease. The


5

isolation of R and Avr genes has been critical for understanding the

underlying molecular mechanisms of race-specific immunity in plants. Many

R genes from monocots and dicots have been cloned during the last 10 years,

encoding R proteins to bacterial, viral, fungal, oomycete, nematode and insect

pathogens (Dangl and Jones, 2001; Hammomd-Kosack and Parker, 2003;

Table 1 for an overview of isolated plant R genes). Products of known Avr

genes encode highly diverse effector molecules that are released during

pathogenesis (Bonas and Lahaye, 2002).

Deduced R proteins from a number of plant species to different

pathogen classes (insects, fungi, bacteria, viruses, oomycetes, nematodes)

revealed striking sequence similarities and a limited number of modular

structural features. This strongly suggests the existence of common molecular

recognition mechanisms in plants to microbial pathogens. Most R genes

encode proteins containing variable numbers of sequence-diversified Leucine-

rich repeats (LRRs), a protein domain that is known to participate in protein-

protein interactions (Jones and Jones, 1996; Kobe and Deisenhofer, 1995;

Kajava, 1998). LRR containing R proteins can be broadly divided into two

classes, one with intracellular and the other with extracellular LRRs (eLRRs;

see below). The largest class of known R genes encodes predicted

intracellular proteins. These share a central nucleotide-binding (NB) site and

C-terminal LRRs. The NB site includes kinase 1a (also called P-loop), kinase

2 and 3a motifs (Traut, 1994) and is part of an extended domain, designated

NB-ARC, which includes additional sequence motifs present in animal cell

death effectors such as APAF-1 and CED4 (NB-ARC is an acronym for a

nucleotide-binding adaptor shared by APAF-1, most known intracellular NB-

LRR plant R proteins, and CED-4; van der Biezen & Jones, 1998; Dangl and

Jones, 2001). Members of this class can be further divided in two subclasses

containing either N-terminal sequences predicted to form a coiled-coil (CC)

structure (CC-NB-LRR subfamily) or sequences that are related to the

cytoplasmic domain of the Drosophila Toll and human interleukin-1 receptor

6

Table1. The major classes of isolated plant resistance genesa

Class R gene Plant Pathogen (Avr gene or product) Predicted structure of R protein

Race-specific

References

1-a

RPS2 RPS5 RPM1 RPP8 HRT Prf Mi-1 I2 Rx1 Rx2 Gpa2 R1 Dm3 Bs2 Xa1 Pib Pi-ta Cre3 Rp1-D Mla1 Mla6

Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Tomato Tomato Tomato Potato Potato Potato Potato Lettuce Pepper Rice Rice Rice Wheat Maize Barley Barley

P. syringae p.v. tomato(avrRpt2) P. syringae p.v. tomato (avrPphB) P. syringae p.v. maculicula (avrRpm1;avrB) Peronospora parasitica (avrRpp8) Turnip crinkle virus (Coat protein) P. syringae p.v. tomato (avrPto) Meloidogyne incognita (? nematode); Marcosiphum euphorbiae (? aphid) Fusarium oxysporum Potato virus X (Coat protein) Potato virux X (Coat protein) Globodera pallida Phytophthora infestans (race1) Bremia lactuca X. campestris p.v. vesicatoria (avrBs2) X. oryzae p.v. oryzae Magnaporthe grisea Magnaporthe grisea (avrPita) Heterodera avenae Puccinia sorghi Blumeria graminis f.sp. hordei (avrMla1) Blumeria graminis f.sp. hordei (avrMla6)

CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRD CC-NB-LRR CC-NB-LRR CC-NB-LRR CC-NB-LRR

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Bent et al., 1994 Warren et al., 1998 Grant et al., 1995 McDowell et al., 1998 Cooley et al., 2000 Salmeron et al., 1994 Milligan et al., 1998 Rossi et al., 1998 Simons et al., 1998 Bendahmane et al., 1999 Bendahmane et al., 2000 Van der Voort et al., 1999 Ballvora et al., 2002 Meyers et al., 1998 Tai et al., 1999 Yoshimura et al., 1998 Wang et al., 1999 Bryan et al., 2000 Lagudah et al., 1997 Collins et al., 1999 Zhou et al., 2001 Halterman et al., 2001

7

1-b

2

3 4 5 6

N RPS4 RPP1,10,14 RPP4,5 L6, L1-12 M RRS-1 Cf-9 Cf-4 Cf-2 Cf-5 Hcr9-4E Hs1pro-1

Ve1 Ve2 Xa21 FLS2 Hm1

Tobacco Arabidopsis Arabidopsis Arabidopsis Flax Flax Arabidopsis Tomato Tomato Tomato Tomato Tomato Sugar beet Tomato Tomato Rice Arabidopsis Maize

Mosaic virus (Replicase) P. syringae p.v. tomato (avrRps4) Peronospora parasitica Peronospora parasitica Melampsora lini (AL6) Melampsora lini (AM) Ralstonia solanacearum (race1) Cladosporium fulvum (avr9) Cladosporium fulvum (avr4) Cladosporium fulvum (avr2) Cladosporium fulvum (avr5) Cladosporium fulvum (avr4E) Heterodera schachtii Verticillium albo-atrum Verticillium albo-atrum X. oryzae p.v. oryzae (all races) Multiple bacteria (flagellin) Cochliobolus carbonum (race1)

TIR-NB-LRR TIR-NB-LRR TIR-NB-LRR TIR-NB-LRR TIR-NB-LRR TIR-NB-LRR

TIR-NB-LRR-NLS-WRKY

eLRR-TM-sCT eLRR-TM-sCT eLRR-TM-sCT eLRR-TM-sCT eLRR-TM-sCT eLRR-TM-sCT

CC-eLRR-TM-ECS

eLRR-TM-PEST-ECS

eLRR-TM-kinase eLRR-TM-kinase

Detoxifying enzyme HC toxin reductase

Yes Yes Yes Yes Yes Yes

Yes

Yes Yes Yes Yes Yes Yes

Yes Yes

Yes No

Yes

Whitham et al., 1996 Gassmann et al., 1999 Botella et al., 1998 Van der Biezen et al.,2002 Lawrence et al., 1995 Anderson et al., 1997 Deslandes et al., 2002 Jones et al., 1994 Thomas et al., 1997 Dixon et al., 1996 Dixon et al., 1998 Takken et al., 1999 Cai et al., 1997 Kawchuk et al., 2001 Kawchuk et al., 2001 Song et al., 1995 Gómez-Gómez et al., 2000 Johal and Briggs, 1992

8

7 8 9

mlo Rpg1 RPW8.1 RPW8.2

Barley Barley Arabidopsis

Blumeria graminis f.sp. hordei Puccinia graminis f.sp. tritici Multiple powdery mildew species

7 TM protein

Receptor kinase-like protein with 2 tandem kinase domains Small, probable membrane protein with CC domain

No

No

No

Bueschges et al., 1997 Brueggeman et al., 2002 Xiao et al., 2001

a Compiled from van’t Slot KAE and Knogge W, 2002; Hammond-Kosack and Parker, 2003; CC, Coil-Coiled domain; ECS, endocytosis signal; LRD, leucine-rich domain; LRR, Leucine-rich repeat; PEST, Pro-Glu-Ser-Thr; sCT, single cytoplasmic tail; TIR, Drosophila Toll and the mammalian interleukin-1 receptor; TM, transmembrane


9

(TIR-NB-LRR subfamily). Most NB-LRR type R proteins consist of these

protein modules except few containing additional domains. For example,

Arabidopsis RRS1-R, confers resistance against the bacterium Ralstonia

solanacearum and possesses an additional C-terminal WRKY domain

(Deslandes et al., 2002). Some NB-LRR R proteins contain an additional C-

terminal non-LRR region (CT region) lacking homology to known protein

domains (Dodds et al, 2001; Shen et al, 2003).

A second eLRR containing R protein class is membrane-anchored by a

single transmembrane helix. Structural variations are also found within

members of this class. For example, the rice Xa21 product has an additional

intracellular Ser/Thr kinase module, whereas the tomato Cf gene products

lack any significant intracellular domains (reviewed by Ellis et al., 2000). Two

more recently isolated R genes from tomato, Ve1 and Ve2, encode eLRR type

proteins with a cytoplasmic domain possessing sequences that in mammalian

receptors stimulate their endocytosis and degradation (the ECS domain;

Kawchuk et al., 2001).

The modular structural organization of plant R proteins might be

significant with regard to distinct functions possibly fulfilled by an individual

domain as well as co-operations among different domains. In plants, the LRR

domain of membrane-anchored Cf proteins has been shown to have a role in

recognition specificity (Van der Hoorn et al., 2001; Wulff et al., 2001). This

was shown by domain swap experiments between sequence-related Cf

proteins recognizing different pathogen-derived effectors. Similar data have

been reported so far only for intracellular TIR-NB-LRR proteins encoded by

alleles of the flax rust R locus L and were shown for the first time in this work

for a CC-NB-LRR-CT type R protein (Ellis et al., 1999; Luck et al., 2000; Shen

et al., 2003).

Unlike highly variable LRR sequences, the NB-ARC domain is largely

conserved among NB-LRR type R proteins. Recently, biochemical analysis


10

and site directed mutagenesis of residues implicated in nucleotide

binding/hydrolysis (kinase1a, 2 and 3a motifs) of the NB-ARC domain of

tomato R proteins I-2 and Mi-2 provided experimental evidence for ATP

binding but not of other nucleotide triphosphates (Tameling et al, 2002). Thin

layer chromatography revealed that both I-2 and Mi-1 exerted ATPase

activity, suggesting that the NB-ARC domain is a functional nucleotide binding

pocket capable of binding and hydrolyzing ATP (Tameling et al., 2002).

Because most characterized R-triggered plant immune responses are tightly

linked to a localized cell death response at sites of attempted pathogen

infection (frequently termed hypersensitive response; HR) and NB-ARC

adaptor containing Caenorhabditis elegans CED-4 and its human homolog

APAF-1 mediate programmed cell death during development (apoptosis), it

has been hypothesized that the NB-ARC domain in plants may have similar

functions in death signalling as in animals (van der Biezen and Jones, 1998a).

Interestingly, separate expression of the CC–NB and LRR parts of the

potato R protein Rx to potato virus X (PVX) resulted in intramolecular physical

interactions in planta (as did the CC domain with the NB–LRR part) and both

interactions were disrupted in the presence of the PVX effector (Moffett et al.,

2002). However, the interaction between the CC and NB–LRR parts was

dependent on a wild-type P-loop motif in the NB-ARC domain, whereas the

interaction between CC–NB and LRR was not (Moffett et al., 2002). It was

concluded that activation of Rx involves sequential disruption of at least two

intramolecular interactions (Moffett et al, 2002). In analogy to APAF-1

function, it has been hypothesized that the activation of R proteins may

involve Avr-dependent release of the NB-ARC domain from inhibition by the

C-terminal LRRs , followed by multimerization of a complex that recruits

additional proteins to the amino-terminal domain for further signalling events

(Dangl and Jones, 2001). It is conceivable that the different N-terminal

structures of cytoplasmic NB-LRR protein, TIR or CC domains, respectively

links to one of at least two distinct signalling pathways specified by different


11

components (Aarts et al, 1998).

1.3. The Mla locus and Mla resistance genes

1.3.1. Molecular characterization of the Mla locus and Mla genes

In barley, R genes to Bgh have been mapped to 10 loci: Mla, Mlat,

MlGa, Mlk, Mlnn, Mlra, Mlp on chromosome 5 (1H; barley chromosome 5 is

also denoted as 1H according to its homoeologous relationships with

chromosomes of other Triticeae species; Barley Genetics Newsletter V27);

Mlg on chromosome 4 (4H); MlLa on chromosome 2 (2H) and Mlh on

chromosome 6 (6H). Out of a total of approximately 85 identified resistance

specificities (Jørgensen 1994; Görg et al., 1993; Büschges et al., 1997; Giese

et al., 1993), approximately 30 are encoded at the Mla locus on the short arm

of chromosome 5. These resistance specificities have been defined using a

large set of differential barley accessions and powdery mildew isolates that

produce gene-for-gene type interactions (Giese, 1981; Giese et al 1981;

Wise and Ellingboe, 1983, 1985; Jahoor and Fischbeck, 1993; Jorgensen

1994). Most of the Mla resistance specificities have been introduced into

barley cultivars from the wild relative Hordeum spontaneum. This suggests

that much of the recent coevolution between barley and Bgh was

concentrated at a single R locus, Mla, in the host. Due to its highly

polymorphic nature, the Mla locus is an excellent model to study ‘gene-for-

gene’ specific recognition events of effectors encoded by a biotrophic fungal

pathogen.

Various molecular marker techniques were used to genetically map the

complex Mla locus. The locus was genetically and physically delimited within

an interval of approximately 240 kb on chromosome 5 ({Wei, 1999 #27}). DNA

markers tightly linked to Mla were used to identify BAC contigs from cultivar

Morex spanning the Mla cluster. A contiguous DNA sequence of the interval in

Morex revealed 32 predicted genes of which eight encode CC-NB-LRR

resistance gene homologs (RGHs; {Wei, 2002 #45}). The RGHs belong to


12

three dissimilar families sharing less than 43% amino acid sequence similarity

between families (Wei et al., 1999; 2002). Since Morex lacks a known Mla

resistance specificity, the first two identified Mla powdery mildew R genes,

Mla1 and Mla6, were isolated from other barley accessions (Halterman et al.,

2001; Zhou et al., 2001). The deduced proteins share 91% identical residues

and show each highest overall similarity to the deduced Morex RGH1bcd

family member (83% and 79% identity to MLA1 and MLA6, respectively)

(Halterman et al., 2001; Wei et al., 2002). Recently two further specificities,

Mla12 and Mla13, have also been isolated (Halterman et al., 2003; part of the

present work). All Mla R specificities isolated to date share a common

exon/intron structure and encode CC-NB-LRR type proteins that possess an

extra C-terminal non-LRR (CT) region (CC-NB-LRR-CT structure). The extent

of sequence similarity between deduced MLA R proteins is remarkable: ~97%

sequence identity in the CC-NB domains and ~87% in the LRR-CT region

(Halterman et al., 2003; Shen et al, 2003).

1.3.2. Mla-mediated infection types

A common feature of most but not all characterized R gene-mediated

resistance responses is a rapid and localized host cell death (HR) at

attempted infection sites that is thought to shut off nutrient supply to microbial

pathogens (Shirasu and Schulze-Lefert, 2000). Although MLA R proteins to

Bgh are highly sequence-related, immune responses triggered by different

Mla R specificities result in diverse infection phenotypes (Boyd et al., 1995).

This was shown by quantitatively assessing Bgh growth stages and the timing

of HR onset at single interaction sites in a set of near-isogenic barley lines

containing different Mla R specificities. To exclude genetic background

variation of different Bgh isolates, Boyd et al. used a single isolate expressing

multiple AvrMla genes. Mla1 and Mla6 resistance terminates fungal growth at

an early stage (essentially no secondary hyphae formation on the leaf

surface) and triggers a rapid HR that is mainly confined to attacked leaf

epidermal cells. In contrast, Mla3 and Mla7 mediate growth cessation at a


13

later developmental stage, permitting growth of some elongating hyphae. This

is linked with a delayed onset of HR including both epidermal and subtending

mesophyll cells (Boyd et al., 1995). Consistent with a rapid Mla1-mediated

resistance response, Koga et al. reported fungal growth cessation coincident

with haustorium maturation and onset of an epidermal HR within 24 hours

after spore inoculation (Koga et al., 1990). Race-specific immunity triggered

by an barley R gene at another R locus to Bgh, Mlg, was shown to terminate

Bgh growth even earlier, i.e. concomitant with the process of cell wall

penetration before onset of haustorium differentiation (Görg et al., 1993).

Many factors could contribute to the phenotypic variation of R gene-triggered

resistance responses to Bgh (see discussion). Mlg gene dosage experiments

(Mlg Mlg, Mlg mlg, mlg mlg genotypes) in a near-isogenic background as well

as greatly different infection phenotypes reported in homozygous and

heterozygous Mla12 lines (Görg et al., 1993; Torp and Jørgensen, 1986)

suggest that R protein levels could be rate-limiting for the onset and/or speed

of resistance responses (if R gene dosage is directly linked to R protein

levels).

1.4. Rar1 and Sgt1--- two genes required for disease resistance

1.4.1. The Rar1 gene

A mutant screening of suppressors of Mla12 function identified barley

Rar1 (Required for Mla12 resistance-1) (Torp and Jørgensen, 1986;

Jørgensen, 1996). The susceptible rar1 mutants are unable to mount an HR

response and also show a significant reduction in the incidence of whole-cell

H2O2 accumulation (Freialdenhoven et al, 1994; Shirasu et al, 1999). Genetic

studies have shown that wild-type Rar1 is required for many, but not all, Mla R

specificities to Bgh (Jørgensen, 1996). In addition, several powdery mildew R

loci on other barley chromosomes require Rar1 for efficient resistance

(Jørgensen, 1988; Freialdenhoven et al, 1994). Similar mutational studies or

virus induced gene silencing (VIGS) experiments revealed that Rar1

homologues in Arabidopsis and Nicotiana benthamiana play a conserved role


14

in the function of a subset of NB-LRR R proteins that confer resistance to

different pathogens, e.g. oomycete, bacteria, fungus and virus (Liu et al.,

2002a; Muskett et al., 2002; Tornero et al., 2002). This revealed that RAR1

activity is essential for the function of both structural R subtypes, TIR–NB–

LRR and CC–NB–LRR proteins.

Barley Rar1 gene was isolated by a map-based cloning approach

(Freialdenhoven et al, 1994; Lahaye, 1998a,b; Shirasu,et al 1999a). The

deduced intracellular 25.5-kD RAR1 protein contains a pair of tandemly

duplicated 60 amino acid sequence-related domains, designated CHORD-I

and CHORD-II (cysteine- and histidine-rich domains), each possibly adopting

a novel zinc-finger structure (Shirasu, et al., 1999). DNA sequence data of

RAR1 homologues and systematic database searches revealed examples

with similar arrangement of CHORD domains from a broad range of phyla in

addition to plant species, except in yeast (reviewed in Shirasu and Schulze-

Lefert, 2003). Although the two CHORD domains show overall sequence

similarity, distinctive sequence features of each domain are conserved across

proteins from different species, suggesting non-identical functions performed

by each CHORD (reviewed in Collins et al, 2003). In plants, an extra stretch of

~20 highly conserved amino acids, termed the CCCH motif, is located

between the CHORD domains, while metazoan RAR1 homologs contain an

extra C-terminal extension adjacent to CHORD-II, designated the CS motif

(CHORD and SGT1 motif).

1.4.2. The Sgt1 gene

Kitagawa et al. (1999) originally identified SGT1 as essential

component for cell cycle progression at G1/S and G2/M transitions in yeast.

Isolation and characterization of temperature sensitive mutant sgt1 alleles

revealed that yeast SGT1 physically associates with SKP1 in at least two

complexes: the CBF3 (centromere binding factor 3) kinetochore complex and

the SCF (SKP1/CULLIN/F-box protein) ubiquitin ligase complex (Kitagawa et


15

al., 1999). SCF complexes play a broad role in regulating the stability/activity

of many proteins in diverse physiological processes, recruit specific

substrates and catalyze their ubiquitination, thereby often marking the

substrates for degradation by the proteasome (Hochstrasser, 2000). The sgt1-

3 mutant protein abolishes the interaction with SKP1 and leads to

compromised CBF3 complex assembly, while ubiquitination of SCF target

proteins remained unaltered in this yeast mutant (Kitagawa et al 1999). In

contrast, the sgt1-5 mutant protein leads to compromised SCF function but

retains its ability to interact with SKP1 and retains also CBF3 function. This

strongly suggests allele-specific perturbations of distinct SGT1 functions and

indicates that the physical association between SGT1 and SKP1 is not critical

for SCF activity.

Yeast two-hybrid screenings for interacting partners of Arabidopsis

RAR1 identified two proteins with significant sequence similarity to yeast

SGT1, designated as AtSgt1a and AtSgt1b (Azevedo et al, 2002). This

protein-protein interaction is conserved since both AtSGT1a and AtSGT1b

were found to interact also with barley RAR1. All known SGT1 proteins in

species from different phyla contain the CS motif that metazoan RAR1

homologs also possess at the C-terminal end (Shirasu et al., 1999; Kitagawa

et al., 1999). This finding is indicative of an ancient CS domain fusion event.

Such fusion events often indicate a functional link between two proteins

mediated by direct protein-protein interactions (Rosetta stone hypothesis;

Marcotte et al., 1999). Co-immunoprecipitation experiments, using barley leaf

protein extracts from non-inoculated plants, corroborated a physical

interaction between SGT1 and RAR1. Furthermore, dsRNAi gene silencing of

Sgt1 showed that Mla6 but not Mla1 requires Sgt1 for full resistance to Bgh

and co-silencing of SGT1 and RAR1 resulted in an additive level of

susceptibility, again indicative of co-operation between these proteins in Mla

gene-mediated resistance (Azevedo et al, 2002). These dsRNAi gene


16

silencing experiments in barley provided genetic evidence for a critical role of

barley Sgt1 as novel factor in Mla-mediated race-specific resistance.

In Arabidopsis, sgt1b mutants were identified in a forward genetic

screen for plants defective in resistance mediated by the R gene, RPP5, to an

isolate of the oomycete pathogen Peronospora parasitica (Austin et al., 2002).

Sgt1b mutants, like Rar1 mutants, exhibit significantly disabled HR and

reduced whole cell H2O2 accumulation at most infection sites, allowing

efficient colonisation of P. parasitica. Interestingly, although a delayed plant

cell death response was observed in both sgt1b and rar1 single mutants

followed by appearance of necrotic plant cells trailing the pathogen at later

stages of infection (trailing necrosis), the double sgt1b/rar1 mutant has

additive disease susceptibility and no plant cell death response was observed.

The conclusion from these data is that SGT1b and RAR1 co-operate in RPP5-

mediated resistance, consistent with the results obtained from experiments in

barley (Azevedo et al, 2002; Austin et al., 2002; reviewed in Muskett and

Parker, 2003).

More genetic evidence supports a more general role of Sgt1 in R-gene

triggered disease resistance in plants. Using a virus-induced gene silencing

approach in Nicotiana benthamiana, silencing of the two copies of SGT1 in

this plant compromised the functions of Rx conferring resistance to potato

virus X (PVX) and tobacco N to the tobacco mosaic virus (TMV) (Peart et al,

2002; Liu et al., 2002b). Interestingly, SGT1 was also found involved in non-

host resistances against certain types of pathogens (Peart et al, 2002). Non-

host resistance is a class of disease resistance in plant species that are

outside the host range of a pathogen species (Heath 2000). Taken together,

SGT1 serves critical roles in R-gene mediated disease resistance to different

pathogen classes as well as in certain non-host responses; like RAR1, SGT1

is required for resistance triggered by R proteins from both TIR-NB-LRR and


17

CC-NB-LRR subclasses, indicating its common role in plant disease

resistance.

1.5. Current models of pathogen recognition in plant disease resistance

1.5.1. Direct physical R protein-AVR effector interaction

A commonly accepted theory regarding pathogen-host plant

interactions is the “gene-for-gene” hypothesis, put forward by Flor more than

50 years ago when he worked with flax and the flax rust fungus (Flor, 1971).

Central to this theory is that disease resistance in plants commonly requires

two complementary genes: an avirulence (Avr) gene in the pathogen and a

matching, resistance (R) gene in the host. One out of several possible

biochemical interpretations of this hypothesis is a receptor-ligand model in

which plants activate defence mechanisms upon R-protein-mediated

recognition of pathogen-derived Avr products (Hammond-Kosack and Jones,

1997). Most plant R proteins contain either an extra- or intracellular LRR

domain that is thought to participate in protein-protein interactions (Kobe and

Deisenhofer, 1994; Kajava 1998). Importantly, sequence comparisons of both

NB-LRR or membrane-anchored type R proteins shows that the predicted

solvent-exposed residues in the LRRs are hypervariable and subject to

diversifying selection (Botella et al., 1998; McDowell et al., 1998; Meyers et

al., 1998; Halterman et al., 2001). This is interpreted as evidence that R

proteins have the capacity to directly recognize pathogen effectors. However,

extensive studies carried out for many Avr-R gene pairs, has shown only two

examples supporting such a direct interaction (the rice blast resistance protein

Pi-ta and Avr-Pita from Magnaporthe grisea, and RRS1-R/PopP2 of

Arabidopsis and Ralstonia solanacearum (Jia et al., 2000; Deslandes et al.,

2003). Thus, it seems possible that at least some R proteins mediate indirect

pathogen recognition by a process involving additional host proteins.

1.5.2. The Guard Hypothesis


18

The 'guard hypothesis' postulates that R proteins function in the

surveillance of a host protein or a complex (the ‘guardee’) that is targeted by

AVR products for modifications favoring pathogen growth. Detection of the

modifications by the R protein triggers the resistance response (Dangl and

Jones, 2001). Initial evidence for this model was found in disease resistance

triggered in tomato plants to the tomato speck pathogen P. syringae

containing AvrPto. The resistance response was shown to require two host

proteins, the NB-LRR protein Prf and the Pto protein kinase; while Pto was

found to interact physically with AvrPto (Scofield et al., 1996; Tang et al.,

1996) Prf does not. Pto is considered to be the virulence target of AvrPto,

which is guarded by the R protein, Prf (Van der Biezen and J.D.G. Jones,

1998b).

More evidence is emerging to support the indirect recognition model.

The study of Arabidopsis-Pseudomonas interactions identified RIN4 (RPM1-

interacting protein) as a common ‘guardee’ targeted by two sequence

unrelated effectors, AvrRpm1 or AvrB (Mackey et al, 2002). RIN4 was first

identified in yeast two-hybrid screens to interact with AvrB, and was

subsequently found to interact also with the NB-LRR type protein RPM1

conferring resistance against Pseudomonas syringae expressing AvrRPM1

and AvrB. RIN4 was shown to co-immunoprecipitate with AvrB, AvrRpm1,

and the NB-LRR protein RPM1 in vivo. RIN4 is essential for RPM1-dependent

defences, as the reduction of RIN4 protein levels inhibits the restriction of

pathogen growth and the HR in response to bacteria that express AvrRpm1 or

AvrB. Phosphorylation of RIN4 was induced by AvrRpm1 and AvrB,

independent of the presence of RPM1. It was proposed that RIN4 positively

regulates RPM1-mediated resistance.

More evidence for the role of RIN4 as a guardee of another NB-LRR

protein, RPS2, was recently described (Mackey et al., 2003; Axtell and

Staskawicz, 2003). In the same pathosystem another R-Avr gene pair


19

(AvrRpt2-RPS2) was used to explore the relationships involving RIN4. RPS2

was shown to physically interact with RIN4. Furthermore, it was found that

AvrRpt2 induces RIN4 disappearance. Over-expression of RIN4 blocks the

detection of AvrRpt2 by RPS2, while loss-of-function rin4 mutations are lethal

in RPS2 plants but have no phenotype in rps2 mutant plants independent of

pathogens. These data provide evidence for interference between two R

protein functions and suggest that RIN4 is a negative regulator of RPS2

function. RPS2 appears to detect the disappearance of RIN4 mediated by

AvrRPT2 and triggers cell death-associated resistance when RIN4 levels drop

below a threshold. Despite these advances it remains unclear which

biochemical role RIN4 serves during Pseudomonas pathogenesis and how

the LRRs of RPS2 and RPM1 participate in pathogen recognition. Moreover,

the present data do not exclude the possibility of transient direct interactions

between R and AVR proteins that might occur subsequent to an initial binding

of the bacterial effectors to the guardee, e.g. through conformational changes

of RIN4 containing heterocomplexes.

The Arabidopsis genome contains approximately 128 and the rice

genome an estimated number of 600 NB-LRR type genes (The Arabidopsis

Initiative, 2000; Dangl and Jones 2001; Goff et al., 2002). Although many R

genes are highly polymorphic in natural populations of a species, are often

organized in R gene clusters, and evolve faster than the rest of the genome,

no experimental evidence exists for a dedicated machinery that facilitates the

generation of new R gene specificities. Therefore, it remains a fundamental

question whether a plant species encodes a sufficient repertoire of R proteins

to directly recognize the collective repertoire of effectors generated by all

pathogenic microorganisms. Unlike the receptor-ligand model, indirect

recognition of effector activities by R proteins may necessitate the presence of

a smaller number of R genes since one would expect a limited number of

effector targets (guardees) in the host that can be manipulated to the

advantage of pathogens.

20

2. Material and Methods

2.1. Materials

2.1.1 Antibiotics

Ampicillin (1000x): 100 mg/ml in H2O

Kanamycin (1000x): 50 mg/ml in H2O

Stock solution stored at –20 oC

2.1.2 Antibodies

Listed below are optimum dilutions for each antibody used in the

present study. The secondary antibodies are all Horseradish

Peroxidase (HRP) labelled.

Antibodies and dilutions

Primary Dilution Secondary Dilution

HA 5,000 Rat IgG 5,000

Myc 1,000 Rabbit IgG 10,000

LexA 500 mouse IgG 10,000

2.1.3 Bacterial strains

2.1.3.1 E coli strains

DH5α:

Genotype: supE44 DlacU169 hsdR17, recA1, endA1, gyrA96,

thi-1, relA1, F-

DH10B:

Genotype: F-, mcrA∆(mrr-hsdRMS-mcrBC)Φ80dlacX74, deoR,

recA1, endA1, araD139, (ara,leu)7607, galU, galK, λ – rspl,

nupG

Material and Methods

21

2.1.4 Yeast strains

EGY48(8Op-LacZ): Yeast strain EGY48 transformed with the

autonomously replicating p8op-lacZ plasmid.

Genotype: MATa, ura3, his3, trp1, LexAop (x6)-LEU2

YM4271

Genotype: MATa, ura3- 52, his3- 200, lys2-801, ade2-101,

ade5, his3, trp1, trp1-901, leu2-3, 112, tyr1-501, gal4∆, gal80∆,

ade5 : : hisG

AH109

Genotype: MATa, trp1-901, leu2-3, 112, ura3-52, his3-200,

HIS3, ADE2, lacZ, trp1, leu2, gal4∆, gal80∆, LYS2 : : GAL1UAS-

GAL1TATA- HIS3, MEL1 GAL2 UAS -GAL2 TATA -ADE2,

URA3::MEL1UAS-MEL1 TATA -lacZ

Y190

Genotype: MATa, ura3- 52, his3- 200, ade2- 101, lys2-801, trp1,

leu2, trp1- 901, leu2- 3, 112, gal4∆, gal80∆, cyhr2, cyhr2, LYS2 :

: GAL1 UAS-HIS3 TATA-HIS3, MEL1, URA3 : : GAL1UAS-GAL1 TATA-

lacZ

2.1.5 Fungal strains

The known avirulence/virulence gene profiles of the two Bgh

isolates is listed below (Avr-Avirulence, vir-virulence)

Isolate A6:

Avr: AvrMla3, AvrMla6, AvrMla9, AvrMla10, AvrMla12, AvrMla13,

AvrMlg, AvrMl(CP), AvrMlH, AvrMlK1, AvrMlLa, AvrMl(Ab)

vir: virMla1, virMla22 Isolate K1:

Avr: AvrMla1, AvrMla3, AvrMla7, AvrMla22, AvrMlLa, AvrMl(Ab)


22

vir: virMla6, virMla9, virMla10, virMla11, virMla12, virMlg, virMl(CP),

virMlH, virM1K, virMlra

Bgh strains were maintained on live barley plants or detached

leaves. A6 was maintained on P01, a near-isogenic line from cv. Pallas

containing Mla1; K1 was maintained on I10, a near-isogenic line of

Ingrid containing Mla12. Plants or detached leaves were kept at 18 oC,

60% relative humidity, and 16 h light/8 h darkness after inoculation with

Bgh conidia spores.

2.1.6 Plant materials

All barley seedlings were grown at 20 oC and 16 h light/8 h

darkness in a protected environment.

Golden Promise: a barley cv. containing no Mla genes

Sultan-5: a chromosome-doubled haploid barley cv. containing Mla12

I10: a near-isogenic line in Ingrid background containing Mla12

Near-isogenic lines in Pallas background:

P01: containing Mla1

P03: containing Mla6 and Mla14

P10: containing Mla12

Mutant lines generated by chemical mutagenesis from Sultan-5 seeds:

M66: Mla12 Mutant

M86: Mla12 Mutant

M22: originally designated as the rar2 mutant; it is actually a Mla12

Mutant (Chapter 2.2, 2.3).

M100: rar1-2 mutant allele

(Torp and Jorgensen, 1986)

Ingrid (mlo3 Rar1): generated by seven backcrosses with cv. Ingrid

Ingrid (mlo29 rar1-2): double mutant, originally isolated from a

re-mutagenized rar1-2 M2 population, this line was used to test

Rar1 dependency of MLA chimeras in this study.


23

2.1.7 Vectors

pGEM-T Promega

pTOPO Invitrogen, Heidelberg

pENTR 4 Enter vector, GATEWAY® compatible, Invitrogen,

Heidelberg

pDONR 201 Invitrogen, Heidelberg

pDEST 32 (BD) Invitrogen, Heidelberg

pBluescript (S/K)+ Stratagene, Heidelberg

pUbi-GFP-Nos Maize-ubiquitin1-promoter :: GFP :: Nos-polyA-

signal,

(Shirasu et al., 1999)

p8op-LacZ reporter vector in LexA system, LacZ under control

lexAop(x8), CLONTECH

pLexA bait vector in LexA system , LexA(1-202) DNA-

BD, CLONTECH

pB42AD prey vector with acidic activator B42, CLONTECH

pAS2-1(M) bait vector in GAL4 system containing GAL4(1-

147) DNA binding domain, modified as

GATEWAY® compatible, containing attB sites,

CLONTECH

pACT2 prey vector from GAL4 system containing

GAL4(768-881) activation domain, CLONTECH

pQSHvRar1-myc generated in the present study by modifying pLexA

vector, leaving out the LexA DNA binding domain

and the Adh promoter driving the expression of

RAR1-myc tagged variant

pRS315-GAL yeast expression vector containing the Gal

promoter with Leu+ autotrophy selection, for the

expression of MLA-HA tagged variants in the

present study.


24

2.1.8 Oligonucleotides

Listed below are primers used in the present study and were

synthesized by Introvigen or Promega

Primers Primer sequence 5’ 3’

Exon-5as AATCGTCATCATGAGCACCTT

M66-s CTGAGATAGGAAAACGGCAGTTT

Mla12BsrDIs1 ACATTGCATCAGATGTGCTCTG

Mla12DNas2 GCTTCCATTGCCTCCCCAACCCT

Mla1EcoRIas1 AAGCGGCCGCGAATTCTAATACTACTAGGACTC

MlaBbSIs TGGGAATAGCATGTCTTCACAG

MlaBsrDIas1 TGATGCAATGTGAGTCGCTCTGG

MlaBsrDIs1 CTGATCCAGAGCGACTCACATTGC

MlaPstIs1 CTTCTGCAGACTGAGTCATCGGCACCTTGC

MlaAgeIas1 TGGCACCGGTGACAATATCCAT

NotIas GCAAGACCGGCAACAGGATTCAA

P10as TCGCAGTGCAGAGAGTTGGCT

P10s AGCCAACTCTCTGCACTGCGA

P12as TCAAACAATATCTGCGTGGCA

P5as CAAGATCCAACACCTCCAAAAACT

P5s AGTTTTTGGAGGTGTTGGATCTT

sh007 CCGATCAAGCTTGGATCCTGATGGATATTGTCACCGGTGCCATTT

sh008 CGCATGCGGCCGCTCAAGCGTAATCTGGAACATCGTATGGGTAGTTCT

CCTCCTCGTCCTCACACAA

sh009 CGCATGCGGCCGCTCAGTTCTCCTCCTCGTCCTCA

sh010 CCGATCAAGCTTGGATCCTGATGGATATTGTCACCGGTGCCATTTCCA

sh011 CGCATGCGGCCGCTTAAGCGTAATCTGGAACATCGTATGGGTAGT

TCTCCTCCTCGCCCTCACACAA

sh012 CGCATGCGGCCGCTCAGTTCTCCTCCTCGCCCTCA

sh013 CGCATGCGGCCGCTCACTCTGTCGCTTCAGCATA


25

Primers Primer sequence 5’ 3’

sh014

sh015

CCGATCAAGCTTGGATCCTGATGCATAAGCATGGGATAGCTCGCATGC

GGCCGCTCACCTTGAAAGAGATGGCATGA

sh016 CCGATCAAGCTTGGATCCTGATGGGGAATAGCATGTCTTCACA

sh017 CGCATGCGGCCGCTCACAATATCTGCGTGGCAGA

sh018 CCGATCAAGCTTGGATCCTGATGCAACGGCTGCTAGTCAT

sh019 CGCATGCGGCCGCTCAAGCGTAATCTGGAACATCGTATGGGTACTCT

GTCGCTTCAG CATA

sh020 CGCATGCGGCCGCTCAAGCGTAATCTGGAACATCGTATGGGTACCTT

GAAAGAGATGGCATGA

sh021 CGCATGCGGCCGCTCAAGCGTAATCTGGAACATCGTATGGGTACAATA

TCTGCGTGGCAGA

sh022 CCGATCAAGCTTGGATCCTGATGAGCCAACTCTCTGCACTGCGA

sh023 CGCATGCGGCCGCTCAAGCGTAATCTGGAACATCGTATGGGTATCGC

AGTGCAGAGAGTTGGC

sh030 GGTCCAGAACCATAACATGTACA

sh031 GTATGTCGTGTACATGTTATGGT

sh032 GGTCCAGAACCATATCAGCTACA

sh033 GTATGTCGTGTAGCTGATATGGT

sh034 CCTCGTCATTGTTCTCGTTCCCTT

sh035 GGTCAGGTCGTTGTCGCACGTATT

sh036 CCTGACCTACAGGAAAGAGTT

sh037 CGTAAA GCGGCCGCTCAATCAACCTGTACGAGGAA

sh038 GCAACGGTCCGAACCTCATAACAACT

sh039 GAAAGCAACCTGACCTACAGGAAAGAG

sh040 GCATGACGCCGAAAACCATTCTT

sh041 GAGACAGCATAGAATAAGTG

sh042 CGTAAAGCGGCCGCTCACCAGAGCTTGTCTTGGCTGT

sh043 CGTAAAGCGGCCGCTCAGGCAGCGTTCATGCTCTCAAG

sh044 CCGATCAAGCTTGGATCCTGATGCACAAGGGTGTCAAGAA

sh045 CCAGCCTCTTGCTGAGTGGAGATG


26

2.1.9 Enzymes

2.1.9.1 Restriction enzymes

Restriction enzymes were purchased from New England Biolabs

(Schwalbach), Boehringer (Mannheim), GIBCO BRL, Pharmacia Biotech

(Braunschweig), and Stratagene (Heidelberg) unless otherwise stated.

10 x buffers for restriction enzymes were companied with the enzymes

and supplied by manufacturers.

2.1.9.2 Nucleic acid modifying enzymes

Standard PCR reactions were performed using homemade Taq

DNA polymerase while for the cloning of the PCR products, pfu, pfx, pwo

or Expand High Fidelity polymerase were used. Modifying enzymes were

listed below and purchased from various sources:

Taq-DNA Polymerase Homemade

Pfu DNA-Polymerase Stratagene (Heidelberg)

Pfx DNA-Polymerase Invitrogen (Heidelberg)

Pwo DNA-Polymerase Roche (Mannheim)

Expand High Fidelity System Roche (Mannheim)

T4 DNA ligase Roche (Mannheim)

T4 Polynucleotide kinase

DNase I, from bovine pancrease

RNase I, from bovine pancrease

Superscript II RT Invitrogen (Heidelberg)

Shrimp alkaline phosphatase Roche (Mannheim)

GATEWAY® -Technology

BP-Clonase Invitrogen (Heidelberg)

LR-Clonase Invitrogen (Heidelberg)

Lysozyme Roche (Mannheim)

2.1.10 Chemicals


27

Laboratory grade chemicals and reagents were purchased from

Roth (Karlsruhe), Serva (Heidelberg), Boehringer (Mannheim), Merck

(Darmstadt), Beckman (München), GIBCO BRL (Neu Isenburg) and

Sigma (Deisenhofen) unless otherwise stated. Filter paper was

obtained from Whatman. Chemicals for yeast culture, transformation

were obtained from Sigma or Merck unless otherwise stated.

2.1.11 Media

Unless otherwise indicated all the media were sterilized by

autoclaving at 121°C for 20 minutes. Heat labile solutions were sterilized

using filter sterilisation units prior to addition of autoclaved components.

For the addition of antibiotics and other heat liable components the

solution or media were cooled down to 55°C.

LB (Lauria Bertani ) Broth

tryptone peptone 1%

yeast extract 0.5%

NaCl 0.5%

Agar plates

1.5-2% agar was added to the above broth.

SOC-Medium (100 ml)

Bacto -tryptone 2.0g

Bacto -yeast extract 0.5g

1M NaCl 1ml

1M KCl 0.25ml

2M Mg2+ stock, filter-sterilized 1ml

2M glucose, filter-sterilized 1ml

Add tryptone, yeast extract, NaCl and KCl to 97ml

distilled water. Stir to dissolve. Autoclave and cool to room

temperature. Just before each use, add 2M Mg2+ stock and 2M

glucose, each to a final concentration of 20mM.


28

SD medium (1 L, 2% glucose or dextrose, pH to 5.8 if necessary)

Yeast Nitrogen Base 6.7 g

Agar(for plate only) 20 g

Drop-out solution (10X) 100 ml

40% glucose 50 ml

H2O 850 ml

Allow medium to cool to ~ 55°C before adding 3-AT,

cycloheximide, additional adenine, or X-gal. If add the sugar

solution before autoclaving, autoclave at 121°C for only 15 min.

YPD (1 L, 2% glucose or dextrose, pH to 6.5 if necessary)

Peptone 20 g

Yeast extract 10 g

Agar (for plate only) 20 g

40% glucose stock 50 ml

H2O 950ml

YPAD

Add to 1L of YPD 15ml of 0.2% Adenine hemisulfate (final

concentration 0.003%)

Galactose/Raffinose SD/X-gal plates (1L)

Prepare SD medium use 725 ml of H2O and do not add

carbon source and not adjust the pH. Autoclave and cool to 55 oC, then add:

40% Galactose 50 ml

40% Raffinose 25 ml

BU salts (10x) 100 ml

20 mg/L X-gal 4 ml

2.1.12 Buffers and solutions


29

2.1.12.1 General buffers and solutions

Sodium acetate, 3 M

NaC2H3O2·3H2O 408 g

H2O 1000 ml

Dissolve sodium acetate trihydrate in 800 ml H2O, adjust

pH to 4.8, 5.0, or 5.2 (as desired) with 3 M acetic acid, add H2O

to 1 L. Filter sterilize.

TE (Tris/EDTA) buffer

10 mM Tris/HCl (pH 8,0, 7,4 or 7,5)

1 mM EDTA (pH 8,0 ) in dH2O

Tris/HCl (1 M)

Tris-Base 121 g

dH2O 1000ml

Dissolve 121 g Tris base in 800 ml, adjust to desired pH

with concentrated HCl, adjust volume to 1 L with H2O, filter

sterilize if necessary, can be stored up to 6 months at 4 oC or at

room temperature.

EDTA (ethylenediaminetetraacetic acid)-stock (0.5 M, pH 8.0)

Na2EDTA 186,1 g

H2O 1000 ml

Dissolve 186.1 g Na2EDTA in 700 ml water, adjust pH to

8.0 with 10 M NaOH (~50 ml; add slowly), add water upto 1 L.

Filter sterilize.

Sodium phosphate buffer (0.1 M)

Solution A: 27.6 g NaH2PO4·H2O per L (0.2 M final) in water.

Solution B: 53.65 g Na2HPO4·7H2O per L (0.2 M) in water.


30

Mix the different volumes of solutions A and B to 100ml

for desired pH, then dilute with water to 200 ml. Filter sterilize if

necessary. Store up to 3 months at room temperature.

SDS (sodium dodecyl sulfate or sodium lauryl sulfate) (20%,w/v)

SDS 20 g

H2O 100 ml

Slightly heat may be necessary to fully dissolve the powder

IPTG stock (0.1M)

1.2 g IPTG add water to 50 ml final volume, Filter-sterilize

and store at 4 oC.

Ethidium bromide stock (10 mg/ml)

ethidium bromide 0.2 g

H2O 20 ml

Stored at 4 oC in dark or in a foil-wrapped bottle. Do not sterilize.

TAE (Tris/acetate/EDTA) buffer (10x)

Tris base 24.2 g

glacial acetic acid 5.71 ml

Na2EDTA·2H2O 3.72 g

Add H2O to 1 L

TBE (Tris/borate/EDTA) buffer (10x)

Tris base 108 g

boric acid 55 g

H2O 960 ml

0.5 M EDTA (pH 8.0) 40 ml

Carbon sources for yeast cultures

40% glucose or Dextrose


31

40% Galactose.

40% Raffinose

Filter sterilized or autoclaved Store at 4°C

10X BU Salts for yeast (1 L, H2O)

70 g Na2HPO4 • 7H2O

30 g NaH2 PO4

Adjust to pH 7, then autoclave and store at room temperature.

X-gal (20 mg/ml in DMF)

Dissolve 5-bromo-4-chloro-3-indolyl-β-D-

galactopyranoside in N,N-dimethylformamide.

Stored in the dark at –20°C.

2.1.12.2 DNA buffers

DNA Gel loading buffer (6x)

bromphenol blue 0.25%(w/v)

xylene cyanol FF 0.25%(w/v)

sucrose 40%(w/v)

or Ficoll 400 15%(w/v)

or glycerol 30%(v/v)

Store at 4oC (room temperature if Ficoll is used).

Sucrose, Ficoll 400, and glycerol are interchangeable in this

recipe.

DNA extraction buffer

100 mM Tris-HCl pH 8.5, 100 mM NaCl, 50 mM EDTA pH

8.0, 2% SDS and 0.1 mg/ml proteinase K (added at the time of

use)

2.1.12.3 Western buffers


32

10x running buffer (1L)

Tris-HCl 30.2g

Glycine 188g

H2O 800ml

SDS 10% 100ml

H2O

2x loading buffer (40ml)

water 5ml

Tris pH 6.8 (1M) 5ml

SDS (10%) 20ml

glycerol 10ml

Bromphenol blue 0.01g

Prior to use, add DTT (20µl DTT (1M) to 80µl loading buffer)

Transfer buffer (1L)

NaPO4 pH 7 1M 15ml

SDS 10% 5ml

Methanol 200ml

H2O add up to 1L

Pre-cool transfer buffer on ice

PBS (phosphate buffered saline solution) 10x (1L)

Na2HPO4 115g

NaH2PO4 29.6g

NaCl 58.4g

H2O add up to 1L

(pH 7.5)

PBS-T

Add Tween-20 (1/1000 v/v) to 1x PBS solution


33

Blocking milk solution

5% (w/v) skim milk powder made with PBS-T solution

2.2 Methods

2.2.1 Nucleic acid manipulations

2.2.1.1 Polymerase chain reaction (PCR) amplification

PCR amplification Puffer, 10x

200mM Tris/HCl (pH 8.4)

500mM KCl

25mM MgCl2

Stock solution is sterilized by autoclaving

Plasmid or genomic PCR (Taq polymerase)

Reaction mix

Reagent Amount per reaction

Template DNA (genomic or plasmid) 20-50 ng

PCR amplification buffer (10x) 1/10 of reaction volume

dNTP mix (dATP, dGTP, dCTP, dTTP) 0.2 mM each

upstream primer (10µM) 0.5 µM

downstream primer (10µM) 0.5 µM

homemade Taq DNA polymerase 2.5 U

Nuclease free water variable

Thermal profile

Stage Temperature (°C)

Time No. of cycles

Initial denaturation 94 2-3 minutes

Denaturation 94 15-30 seconds

Annealing 50-65 °C 20-60 seconds 25-35 x

Extension 72 1-2 min

Final extension 72 7 min


34

Yeast colonies PCR:

Essentially follow the plasmid or genomic PCR protocol except

that 2 µl of the clear lysate from yeast colony lysised in 25µl of 20 mM

NaOH was used as template and the cycle number was increased to

40x.

PCR with other polymerase, e.g., Pfu, Pfx Pwo, or Expand High

Fidelity System were performed according to the manufacturer’s

protocol.

2.2.1.2 Restriction endonuclease digestion of DNA

All restriction digests were carried using the manufacturers

recommended conditions. Typically, reactions were carried out in 1.5

ml eppendorfs using 1-2 Units of restriction enzyme per 10-20µl

reaction. All digests were carried out at the appropriate temperature in

incubators with proper temperature for a minimum of 30 minutes.

Eppendorfs occasionally were replaced with sterile 250µl PCR tubes

and digests might be carried out in a thermal cycler with a heated lid.

2.2.2 DNA analysis

2.2.2.1. Plasmid DNA isolations

Plasmid DNA was isolated by alkaline lysis method (Birnboim

and Doly, 1979). High quality DNA for single-cell transient assay or

sequencing was isolated using Qiagen or MACHEREY-NAGEL(MN)

Mini-, Midi- or Maxi-prep kit.

Barley cDNA library DNA was isolated combining the alkaline

lysis method and CsCl gradient ultra-centrifugation method. Isolation of

library plasmid DNA from E.coli stock was performed according to

normal max prep method upto the clarification of bacterial lysates.

Afterwards, the lysates were directly precipitated with isopropanol

instead of using cartridge or column with silica membrane for binding


35

DNA. The precipitated DNA was resuspended in TE and further

purified using CsCl gradient ultra-centrifugation method (Sambrook, et

al., 1989). Purified library DNA were tested for concentration and

diluted in TE at ~1µg/µl and stored at –20 °C as aliquots.

2.2.2.2. Plant genomic DNA isolation

The Nucleon PhxtoPure resin system (Amersham LIFE

SCIENCE) was used for DNA isolation from barley leaf materials

according to the manufacturer’s protocol with small modifications.

2.2.2.3. Isolation of DNA fragment from Agrose-gel

The Nucleospin Extract-Kit (MACHEREY-NAGEL) was used to

extract DNA fragments from the agrose-gel according to the

manufacturer’s protocol.

2.2.2.4. DNA sequencing

DNA sequences were determined by the Automatische DNA-

Isolierung und Sequenzierung (ADIS-Unit) in MPIZ on Applied

Biosystems (Weiterstadt, Germany) Abi Prism 377 and 3700

sequencers using Big Dye-terminator chemistry (Sanger et al.1997).

PCR products were purified with the Nucleospin Extract-Kit

(MACHEREY-NAGEL) or Qiagen Extract Kit, ensuring sufficient

amount at appropriate concentration to be directly sequenced. When

large scale of PCR products needed to be purified for sequencing, the

Milllipore Montage™ PCRµ96 filter plate were used, or purified by the

ADIS-Unit by Sephadex method.

2.2.2.5. DNA sequence analysis

Sequencing data were analysed mainly using Clone Manager 6,

version 6.00 and alignment made using Align Plus 4, version 4.10 from

Scientific & Educational Software. Alternatively using the GCG-


36

Programm (Version 10.0) from Genetics-Computer-Group, Inc.,

University of Wisconsin, Madison, or ClustalW

(http://www.ebi.ac.uk/clustalw/).

2.2.2.6. Database searching

DNA sequence data was directly used for database searching

using NCBI Blast (http://www.ncbi.nlm.nih.gov/BLAST/), or translated

into polypeptide for motif similarity searching. Other databases were

used, including Phytopathogenic Fungi and Oomycete EST Database

(Version1.4) (http://cogeme.ex.ac.uk/), TAIR

(http://www.Arabidopsis.org/), TIGR (http://www.tigr.org), IPK Barley

ESTs Database (http://pgrc.ipk-gatersleben.de/), and so on.

2.2.3. RNA analysis

2.2.3.1. Isolation of total RNA from plant tissues

Plant materials were finely ground in liquid nitrogen and

resuspended in the total RNA extraction buffer and incubated at 37°C

for 1 hour. Following three phenol/chloroform extractions, RNA was

precipitated with 1 volume 8 M LiCl prepared in DEPC

(Diethylpolycarbonate) water, washed with 70% ethanol and

resuspended in DEPC treated water.

Alternatively, harvested plant material, previously maintained at

-80°C was transferred to a pre-chilled, autoclaved mortar, then ground

in the presence of liquid nitrogen to a fine powder. Approximately 0.5ml

of tissue was transferred to an RNase-free 2ml centrifuge tube, before

1ml of Tri reagent (Sigma) was added. The sample was vortexed for 10

seconds then placed on dry ice to allow any remaining samples to be

processed. All homogenised samples were left at room temperature for


37

10 minutes. 200µl of chloroform was subsequently added, vortexed for

15 seconds and allowed to stand for 2-15 minutes at room

temperature. The samples were then spun for 20 minutes, at

1,3000rpm and 4°C, in a bench top centrifuge. The upper aqueous

phase was carefully transferred to a fresh RNase free 2ml centrifuge

tube. The RNA was precipitated by adding 500µl of isopropanol, mixing

well and leaving at room temperature for 10 minutes. Centrifuging then

at 1,3000rpm for 10-15 minutes and 4°C helped pellet the RNA. The

supernatant was removed and the white pellet was washed with 1ml of

75% ethanol (absolute ethanol diluted with DEPC treated water 1:3).

The samples were briefly vortexed to dislodge the pellet and

centrifuged again for 10 minutes at 4°C and 1,3000rpm. The

supernatant was removed and the pellet air-dried for 10 minutes. The

RNA was re-suspended in 40-60µl of DEPC water.

2.2.3.2. RT-PCR

Reverse transcription – polymerase chain reactions (RT-PCR)

were carried out by two-steps methods. Using RT superscript II for the

first strand cDNA synthesis by combining 2µg template total RNA, 2µl

10µM oligo dT-18, sample were incubated at 70°C for 10 minutes

before immediately cooling on ice. Subsequently the reaction was

made up to 20µl by adding the following components: 4µl 5× first strand

buffer (250mM Tris pH 8.3/375mM KCl/15mM MgCl2), 2µl 0.1M DTT,

1µl 10mM dNTPs mix and proper amount of DEPC treated water. The

mix was incubated at 42°C for 2 min before add into 1µl (200u) RT

Superscript II. Subsequently, proceed at temperature 25(10’)-42(50’)-

70(15’to inactivate the enzyme) for indicated time.

For subsequent normal PCR, use 2 µl of above mixture as

template, 2.5 µl of DMSO added for 50 µl of reaction volume before

PCR (for disrupting the secondary structure of the single DNA strand).


38

2.2.4. Protein analysis

2.2.4.1. Denaturing SDS-polyacrylamide gel electrophoresis

All denaturing SDS-polyacrylamide gel electrophoresis (SDS-

PAGE) was carried out using the Mini-blot Protean system (BioRad).

Gel preparation

Different percentage gels were used depending on the size of

the protein that was to be resolved. All gels were made fresh on the

day of use. The resolving gel was poured between two glass plates

then overlaid with 2mm of isopropanol. The gel was allowed to set for a

minimum of 25 minutes. Isopropanol was removed and washed using

water, and a stacking gel was poured onto the top of the resolving gel.

A comb was inserted, ensuring no bubbles were trapped and the whole

gel left to set for at least 25 minutes.

Reagents and amount used for different percentage resolving gels

Volume for different percentages of gels (in ml)d

Resolving gel componentsa

7% 10% 12%

H2O 5.5 4 3 30% acrylamide mixb 3.5 5 6

1M Tris-HCl (pH8.8) 5.7 5.7 5.7

10% SDS 0.15 0.15 0.15

10% ammonium persulfatec 0.15 0.15 0.15

TEMED 0.01 0.006 0.006


39

Component volume (in ml)

Stacking gel componentsa

5ml 10ml H2O 2.7 6.8 30% acrylamide mixb

0.67 1.660 1.5M Tris-HCl (pH8.8) 0.5 1.26 10% SDS 0.04 0.1 10% ammonium persulfatec

0.04 0.1 TEMED 0.004 0.01 aAdd in stated order, mixing between subsequent additions. b30% Acrylamide/Bis solution 37.5:1. cMake-up fresh before use. dRecipes prepare solution sufficient for two gels, 1.5mm thick or four gels , 0.75mm thick (7 × 10cm).

Yeast crude protein extraction

Overnight yeast cultures raised in SD selection media and 3

OD600 units of cell pelletes obtained from each culture by centrifugation

at 3500 rpm. Immediately the pelletes were frozen in liquid N2 and

samples were boiled for 5 min, these treatments were repeated for at

least 3 times. Directly 200µl of 2x loading buffer with freshly added DTT

was mixed with samples that can be stored at –20 until use. Samples

need to be boiled for 5 min and centrifuged for 5 min at 13,000 rpm

before 20 µl of supernatants loaded on gel for Western blotting.

Western blot

Proteins resolved on acrylamide gels were transferred to

Hybond – ECL (nitrocellulose) membrane (Amersham pharmacia

biotech) after being released from the glass plates and having their

stacking gel removed with a scalpel. The electroblot apparatus (Mini-

blot Protean III; BioRad) was assembled. The ECL membrane was pre-

equilibrated by immersing in transfer buffer. Transfer was carried out

either at 40mA overnight or 250mA for 1 hour 30 minutes at 4°C.


40

The transfer cassette was dismantled and the membrane

washed 5 min with water, then stain with Poncean (1:20 v/v,

stain:water) for 15 seconds to check for equal loading before rinsing in

excess volumes of water. The membrane was then washed with PBS-T

for 5 min and left to block at room temperature in blocking buffer for 2

hours on a rotary shaker. The blocking solution was removed and the

membrane washed briefly in PBS before the addition of the primary

antibody. The optimum dilution of a particular primary antibody was

determined beforehand. The membrane was incubated in the presence

of the primary antibody for approximately 1 hour 30 minutes at room

temperature on a rotary shaker. The membrane was briefly rinsed in

PBS-T, then washed with excess PBS-T for 3 × 5 minutes.

A horseradish peroxidase (HRP) chemiluminescence system

was used to detect bound antigen/primary antibody conjugates. A

suitable secondary antibody (anti-IgG) was added at an optimised

dilution of between 1:5000 and 1:15000, in PBS or blocking milk

solution. The membrane was incubated at room temperature for a

maximum of 1 hour on a rotary shaker, rinsed briefly in PBS-T, then

washed with excess PBS-T for 3 × 5 minutes. Each membrane was

developed using ECL detection reagents according to manufacturer’s

protocol. Any signal was detected by exposing the membrane to film

(Hyperfilm ; Amersham pharmacia biotech) from 1/2 min to 1 hour.

2.2.5 Transformation of E. coli

2.2.5.1 Preparation of electro-competent E. coli cells

10 ml of an overnight culture of E. coli strain (DH5α) was added

to 1 litre of LB broth and shaken at 37°C until the bacterial growth

reached an OD= 0.5-0.6. The bacteria were pelleted at 5000 x g for 20

minutes at 4°C and the pellet gently resuspended in ice-cold sterile

water. The cells were pelleted as before and again resuspended in ice-

cold water. The process was repeated twice. Finally the cells were


41

gently resuspended in a 1/100 volume of the initial culture in 10%

sterile glycerol, pelleted once more and then resuspended in 5 ml 10%

glycerol. 50 µl aliquots of cells were frozen in liquid nitrogen and stored

at –80 till use.

2.2.5.2 Transformation of electro-competent E. coli cells

20 to 50 ng of salt-free ligated plasmid DNA (or ~1µl of ligated

mix from 10 µl ligation system) was mixed with 50 µl of electro-

competent cells, and transferred to the 1mm cold BioRad

electroporation cuvette. The BioRad gene pulse apparatus was set to

25 µF capacitance, 1.8 kV voltage and the pulse controller to 200

ohms. The cells were pulsed once at the above settings for a few

seconds and 500 µl of SOC medium was immediately added to the

cuvette and the cells were quickly resuspended and incubated at 37°C

for 1 hour. A fraction (~150-300µl) of the transformation mixture was

plated out onto selection media plates.

2.2.6 High-efficiency transformation of yeast competent cells

The protocol

(modified from Gietz, R.D. and R.A. Woods, 2002)

1. Start an overnight culture in YPAD (15 ml) supplemented with

antibiotics at 30 °C

2. Start a new culture in 100 ml YPAD using 5 x108 cells in total and

grow for 5-6 hours at 30 °C (no more than 6 hr).

3. Centrifuge 3500 rpm for 3 min.

4. Resuspend the cells in 25 ml of sterile water and centrifuge again.

5. Resupsend the cells in 1 mL of 100 mM LiAc (freshly made from a

1M stock)

6. Centrifuge at 6000 rpm for 30sec.

7. Resuspend the cells in 500 µl of 100 mM LiAc.


42

8. For 1 transformation Use 50-100 ul of cells and spin down (15 s,

6000 rpm). Remove the supernatant, then add 1 µg of plasmid DNA

diluted in water as 5ul. Vortex at low speed 2sec and, under

vortexing, add 300 ul of Transformation Mix. Vortex for 5 more

seconds.

9. Incubate at 30 °C for 30min. Invert tubes every 10 min.

10. Incubate at 42°C for 45min (time various on strains). Invert tubes

every 10 min.

11. Centrifuge 6000 rpm, 10-15 second.

12. Eliminate supernatant. Add carefully 200 uL of sterile water, set for

10-30 min at RT. Gently resuspend the cells by inverting the tubes.

Plate a dilution of the transformed mixture on selective plate to

estimate the number of transformants. Plate the rest of the

transformed mixture on selection plates.

Yeast Transformation Mix

Times x1 x 5 x 10

PEG 50% 680 3400 6800

1M LiAc 100 500 1000Carrier DNA 140 700 1400water 80 400 800

Library DNA transformation into strain YM4271 (MATα)

Several independent transformations were carried out according

to above protocol, and transformants were selected on ~50 big plates

(Φ145mm) with SD/-Trp agar and incubate for 2-3 days at 30 °C until

colonies appears with size of the tip of a pin. All colonies were

collected with chilled YPAD broth and stored as many 15 ml falcon

aliquots with glycerol at 25% final concentration. In total, ~2x106

independent transformants were obtained.

2.2.7 Yeast two-hybrid screening via interaction mating methods


43

Briefly, overnight bait cultures were raised in proper SD media

containing 4% glucose, and the frozen library aliquots were re-

generated in 10-20 times volume of pre-warmed YPAD for 20 min. 20

OD of each bait culture was mixed with ~10 OD of library cells and

subjected to mating for 4.5 hr in YPAD/PEG solution. Inducing media

was used for the expression of the prey library fusion and incubate for

~6 h, cells were collected and diluted to ≤ 5 OD before plating ~6x106

diploid cells onto selection inducing plates. Plates were incubated for

2-5 days for checking of appearing of colonies (Fig. 12 for overview;

described in Kolonin et al (2000)).

2.2.8 Single-cell transient assay in barley epidermal cells using particle bombardment

Overview

A reporter plasmid containing Mlo and GUS genes (GUS gene

alone in the case of Mlo genetic background), and the respective

effector plasmids were mixed prior to the coating of particles (molar

ratio 2:1, respectively, maximum 5µg DNA). The bombarded leaves

were transferred onto 1% agar plates supplemented with 85 µM

Benzimidazol and incubated at 18 oC for 15 h before high-density

inoculation with Bgh spores. Leaves were stained for GUS and single

leaf epidermal cells attacked by Bgh germlings were evaluated under

the microscope at 48 h after spore inoculation. In dsRNAi single-cell

silencing experiments, particles were co-coated with a construct

encoding an intron-spliced dsRNAi construct targeting HvRAR1 or

HvSGT1 according to Azevedo et al., (2002) (molar ratio 1:1:1, 5µg

total DNA). In the gene silencing experiments the bombarded leaves

were incubated at 18 °C for 48 or 96 h before high-density inoculation

to allow turnover of preformed RAR1 or SGT1.

Particle Delivery System: Biolistic-PDS-1000/He (BIO-RAD)


44

Material preparations

Plant material: one-week old barley plants grew in

phytochamber under controlled conditions

Sucrose and Benzimidazol agar (1-1.5%) plates

Gold particles (0.9-1.0 µm): washed and coated with a reporter

and effector constructs

Spermidine solution (0.1M); CaCl2 solution (2.5M); ethanol

(70% and pure), glycerol (50% in water)

Particle bombardment

Use rupture disc (900psi), apply vacuum up to 27 inch, trigger

shooting

Fungal inoculation

Dusting off high-density fresh Bgh conidium spores on

bombarded leaves

GUS staining

Infiltration with GUS staining solution into bombarded leaves in

falcon, leave for at least 10 h at 37 °C

GUS destaining and fixing

Remove GUS staining solution and add in destaining solution

Microscopy

Use coomassie solution to stain fungal surface structure

and evaluate haustorium index (%) under light microscopy.

Other materials required and recipes


45

Consumables

Macrocarrier

Rupture disc

Gold particles

Hepta adapter (including browser, macrocarier holder, stoping

screen holder)

Gus staining solution (1L)

1M Na2HPO4 57.7 ml

1M NaH2PO4 42.3 ml

0.5M Na2EDTA 20.0 ml

K4Fe[CN6] 2.112 g

K3Fe[CN6] 1.646 g

Triton X-100 (v/v) 0.1%

methanol (v/v) 20%

X-gluc 1 g

X-gluc: 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid,

cyclohexylammonium salt, from Roth

Destaining solution

stock solution

50% glycerol

25% lactic acid

H2O

work solution

stock solution : ethanol (v/v) = 1 : 2

Coomassie solution

coomassie (w/v) 0.6%

methanol (or ethanol)

coomassie: Serva Blue R, from Serva

46

3. Isolation and characterization of a new Mla resistance specificity: Mla12

3.1. Introduction

Mla12 is one of the Bgh resistance specificities that was mapped to the

Mla locus on barley chromosome 5S (1HS; Schwarz et al., 1999). A

cytological comparison of near-isogenic Mla12 resistant and mla12

susceptible barley lines in the genetic background of cultivar Pallas revealed

in the resistant line an epidermal cell death response concomitant with the

establishment of Bgh haustoria (Görg et al., 1993). Similarly, a time course

analysis of cytological events during Mla12-mediated resistance in cv.

Sultan 5 showed host cell death at later stages during fungal pathogenesis

(Freialdenhoven et al., 1994). In this case, fungal growth proceeded within 36

h after Bgh spore inoculation to the stage of identifiable differentiated

haustoria at 60% of interaction sites. Cell death of attacked epidermal cells,

measured by whole-cell autofluorescence, reached a maximum at

approximately 48 h (60% interaction sites; Freialdenhoven et al., 1994).

These data demonstrate that the timing of Mla12-triggered cell death relative

to Bgh growth stage is similar though not identical in different host genetic

backgrounds, terminating fungal growth after the complex process of

epidermal cell wall penetration. It is possible though speculative that this ‘post

penetration resistance’ reflects a relatively late release of the cognate

AvrMla12 effector during pathogenesis, e.g. coincident with haustorium

differentiation.

The availability of susceptible mla12 mutants greatly facilitated the

molecular isolation of Mla12. Sultan 5 seeds were chemically mutagenized by

EMS or NaN3 and 25 susceptible mutants were recovered from M2

Mla12: a new specificity

47

populations (Torp and Jørgensen, 1986; Jørgensen, 1988). Genetic analysis

(after crosses with the resistant Sultan 5 and a line lacking Mla12) indicated

that susceptibility in mutants M66 and M86 is likely due to mutations in Mla12,

whereas susceptibility in another line, M22, possibly resulted from extragenic

suppressor mutations of Mla12 function (Jørgensen, 1988, 1996). The gene

required for Mla12 function was designated Rar2 (Freialdenhoven et al.,

1994). However, a closer investigation of the original data from the genetic

analysis of mutant M22 (Jørgensen, 1988; Freialdenhoven et al., 1994)

suggested another possibility: a mutation in Mla12 leading to a partially

susceptible phenotype.

To isolate Mla12, a genomic cosmid library was constructed using DNA

from cv Sultan 5. The library was screened with a DNA probe corresponding

to the LRR region of MLA1 (Zhou et al., 2001), a known polymorphic region in

NB-LRR type genes. By using a DNA probe representing the LRR region of

MLA1, it was expected to isolate from cv. Sultan 5 only genes that are highly

sequence related to Mla1 rather than any RGH. Sixteen cosmid clones were

isolated from the cosmid library. Low-pass DNA sequencing of the cosmid

clones revealed that all of them contain NB-LRR-type RGHs. Two clones,

designated Sp14-1 and Sp14-4, contain identical RGHs showing ~90%

sequence identity to Mla1 and Mla6 in deduced exon and intron sequences. A

closer comparison of the NB-LRR gene in Sp14-4 with Mla1 and Mla6

revealed an identical 5' untranslated small open reading frame of nine amino

acids and the same intron-exon structure (Halterman et al., 2001; Zhou et al.,

2001). These genes share a simple sequence repeat (AT)n in intron 3,

although the exact numbers of the repeats differ (see Fig. 15; below).

Therefore, the RGH in Sp14-4 was considered a candidate Mla12 gene that

encodes a predicted CC-NB-LRR-CT protein of 108 kD sharing 89% identical

residues with MLA1 and 92% identical residues with MLA6 (Fig. 2).

3.2. Characterization of susceptible mla12 mutant alleles

To confirm the identity of the putative Mla12, genomic DNA was


48

MLA1 1 MDIVTGAISNLIPKLGELLTEEFKLHKGVKKNIEDLGKELESMNAALIKIGEVPREQLDSQDKLWADEVRELSYVIEDVVDKFLVQVDGIQFDDNNNKFKGFMKRTTELLMLA6 1 ...........................................................................................S.........L........MLA12 1 ..........................................................................................KS.........L........ NB-ARC MLA1 111 KKVKHKHGIAHAIKDIQEQLQKVADRRDRNKVFVPHPTRTIAIDPCLRALYAEATELVGIYGKRDQDLMRLLSMEGDDASNKRLKKVSIVGFGGLGKTTLARAVYEKIKGMLA6 111 .......................................P......................................................................MLA12 111 ..................................................................G........................................... MLA1 221 DFDCRAFVPVGQNPHMKKVLRDILIDLGNPHSDLAMLDANQLIKKLREFLENKRYLVIIDDIWDEKLWEGINFAFSNRNNLGSRLITTTRIVSVSNSCCSSHGDSVYQMEMLA6 221 ..............D...............................H......................................................D........MLA12 221 ..............D...............................H......................................................D........ MLA1 331 PLSVDDSRILFWKRIFPDENGCLNEFEQVSRDILKKCGGVPLAIITIASALAGDQKMKPKCEWDILLQSLGSGLTEDNSLEEMRRILSFSYSNLPSHLKTCLLYLCIYPEMLA6 331 ........M..S..........I............................................R......................................V...MLA12 331 ........M..Y........A.I............................................R............................N.........V... MLA1 441 DSKIHRDELIWKWVAEGFVHHENQGNSLYLLGLNYFNQLINRSMIQPIYGFNDEVYVCRVHDMVLDLICNLSREAKFVNLLDGSGNSMSSQGNCRRLSLQKRNEDHQAKPMLA6 441 ..M.S..K.........................................NYSG.A.A...............Y..........T.......S...............VR.MLA12 441 ..M.S..K.........................................NYSG.A.A..........................T.......S................R. LRR MLA1 551 ITDIKSMSRVRSITIFPPAIEVMPSLSRFDVLRVLDLSRCNLGENSSLQLNLKDVGHLTHLRYLGLEGTNISKLPAEIGKLQFLEVLDLGNNHNLKELPSTVCNFRRLIYMLA6 551 F................S..........................................................................R.I...............MLA12 551 LI...........................E..C.....K.....D...........Q.IQ.......C.......T....L...........P........IR....... L R (631) M66 MLA1 661 LNLFGCPVVPPVGVLQNLTSIEVLRGILVSVNIIAQELGNLERLRVLDICFRDGSLDLYKDFVKSLCNLHHIESLRIECNSRETSSFELVDLLGERWVPPVHFREFVSSMMLA6 661 ...V..Q......L.....A..........L........K.KSM.E.E.R.N.......EG..............I.G..........VM............L...E...MLA12 661 ...V..Q.I....................YL............V.D.E.R.N.......EGL.N...........N.R..PG.......M...E........L...K.F. CT MLA1 771 PSQLSALRGWIKRDPSHLSNLSELILSSVKDVQQDDVEIIGGLLCLRRLFIITSTDQTQRLLVIRADGFRCTVDFRLDCGSATQILFEPGALPRAVRVWFSLGVRVTKEDMLA6 771 ......................D.V.P-..E.............A....W-.K.NH........PV...H.I...Q...................ES.VI......A...MLA12 771 ...........Q............T.WP................S....W.VK.IH..............SM.E.....................ES.VI......A... E K (866) M866 MLA1 881 GNRGFDLGLQ-GNLFSLREFVSVYMYCGGARVGEAKEAEAAVRRALEAHPSHPRIYIQMRPHIAKGAHDDDLCEDEEEN-- MLA6 879 ..........-...L...RH.F.LI.............K..L...Q....D.L....D...C..E.........G....-- MLA12 881 ..........EAKDV...WD.F.LL..........K..............R......D...D.QE.........N.D.GEN K M (916) M22

CC

Figure 2. Amino acid sequence alignment of deduced products of the Mla1,

Mla6, and Mla12 genes.

Residues identical to those in MLA1 are shown as dots, and deletions are shown as

hyphens. A predicted CC structure is underlined. The starts of the NB, LRR, and CT

regions are indicated with arrows and are operational according to Zhou et al. (2001).

Boldface letters in the NB-ARC domain indicate amino acid motifs conserved among

known NB-LRR proteins. Boxes indicate amino acid exchanges identified in three

susceptible Mla12 mutants, and affected residues are shaded in black.


49

isolated from the Mla12 mutants M86 and M66 and the putative rar2 mutant

M22. The DNA was used as a template for PCR amplification of the respective

Mla12 mutant alleles. Mla12-specific primers were designed based on the

sequence alignment of known Mla1, Mla6, Mla1-2, RGH1a, and the putative

Mla12. PCR products were purified and then sequenced directly.

DNA sequence analysis of the candidate Mla12 in the susceptible

mutants M66 and M86 revealed in each a single nucleotide substitution

compared with the wild-type gene derived from the cosmid clone Sp14-4. The

substitutions replace amino acid Leu631 with Arg in the second LRR of the

deduced candidate MLA12 protein in M66 and amino acid Glu866 with Lys in

the CT region in M86, respectively (Fig. 2). Therefore, the Sp14-4–derived

candidate gene probably is Mla12.

DNA sequence analysis of the candidate Mla12 in M22 plants revealed

a single nucleotide substitution that replaces amino acid Lys916 with Met in the

CT region (Fig. 2). Previous DNA marker-based mapping of susceptibility

conferred by the M22 mutant revealed its location on chromosome 5(1H) at

the Mla locus between restriction fragment length polymorphism markers

MWG036 and MWG068 (Schüller et al., 1992). This finding suggested that

susceptibility might be caused by a mutation in Mla12 or in a tightly linked

gene. The intragenic single amino acid replacement further suggests that

M22, like M66 and M86, likely is a mutant allele of Mla12 (see below).

3.3. Over-expression of Mla12 alters the resistance kinetics but retains

Rar1 dependence

3.3.1. Over-expression of Mla12 alters the resistance kinetics

To test directly the function of the candidate Mla12 gene, Sp14-4 DNA

was delivered into epidermal cells of detached barley leaves by particle

bombardment. Transformed cells were tested for their ability to activate race-

specific powdery mildew resistance upon inoculation with Bgh conidiospores


50

of isolates expressing or lacking AvrMla12 (isolate A6 harbouring AvrMla6 and

AvrMla12 and isolate K1 harbouring AvrMla1) (Zhou et al., 2001). Infection

phenotypes of transgene-expressing epidermal cells were microscopically

inspected at 48 h after inoculation by scoring the presence or absence of

intracellular Bgh haustoria at single interaction sites. Unlike control

bombardments with cosmid DNA harbouring Mla1 or Mla6, which are known

to mediate race-specific resistance in the transient gene expression assay

(Halterman et al., 2001; Zhou et al., 2001), delivery of Sp14-4 DNA failed to

trigger detectable resistance upon inoculation with Bgh strains A6 and K1

(data not shown). This effect may be caused by insufficient 5' flanking

regulatory sequences (~400 bp upstream of the transcription start) in cosmid

Sp14-4, driving expression of the candidate Mla12, or delayed activation of

Mla12 compared with Mla1 and Mla6 resistance.

To examine this possibility further, the coding region of the Mla12

candidate was subcloned under the control of the strong maize ubiquitin

promoter and the nopaline synthase (Nos) terminator. DNA of this over-

expression construct and two similar control over-expression plasmids

harbouring Mla1 or Mla6 were delivered into leaf epidermal cells of barley cv

Ingrid lacking Mla12 and Mlo (Fig. 3A). Delivery of each plasmid DNA together

with an Mlo-expressing construct resulted in a haustorium index of 2 to 5%

upon challenge with the Bgh isolate containing the cognate Avr genes,

whereas the control compatible interactions showed an index of ~80%. Note

that the very high level of haustorium incidence found in the compatible

interactions likely is the result of co-bombardment of the race-nonspecific

defense modulator Mlo, which renders transformed epidermal cells super-

susceptible to the fungus (Kim et al., 2002). These data provided evidence

that the candidate Mla12 gene subcloned from cosmid Sp14-4 triggered

AvrMla12-dependent Bgh growth termination. Interestingly, bombardments

with empty vector DNA into epidermal cells of cv. Sultan 5, which contains

Mla12, resulted in a high haustorium index of 45% when inoculated with the

incompatible isolate Bgh A6 (Fig. 3B). This finding suggests that Mla12


51

CB

B gh AvrM la 6, AvrM la1 2 A6 ( )

B gh A vrM la 1 K 1 ( )

A

100

100

80

60

40

20

0

20

40

60

80

Haust

orium i

ndex in

GUS e

xpress

ingepi

derma

l cells

(%)

Tran sgene

Ge ne ticba ckgro und ( )mlo-3,R ar1

M la12M lo

M la6M lo

Ingri d

M la1M lo

(L631R)

M lo,Rar1,M la12

M la12Em ptyvector

M 66 (

K916M )M lo,Rar1,

M la12

M la12Em ptyvector

M 22(

)Mlo,rar1-2,

M la12

M la6 M la12Em ptyvector

M 10 0(

)Mlo,Rar1,

M la12

M la6 M la12Em ptyvector

Su lta n5

Figure 3. Complementation of susceptible Mla12 mutants by overexpression of

Mla12 resistance.

Relative single cell resistance/susceptibility upon delivery of various Mla transgenes at 48 h after spore

inoculation is indicated by haustorium indices of attacked β-glucuronidase (GUS)–expressing cells (%).

Data shown were obtained by bombardment of plasmid DNAs into epidermal cells of detached barley

leaves (described by Shirasu et al., 1999b; Zhou et al., 2001). A β-glucuronidase reporter gene was

used to identify transformed cells.

(A) The indicated transgenes were tested in detached leaves of barley cv Ingrid harboring mlo-3 Rar1.

In this line, broad-spectrum mlo-3 resistance was complemented by cobombardment with a plasmid

expressing wild-type Mlo; this renders cells supersusceptible to all tested Bgh isolates (Zhou et al.,

2001; Kim et al., 2002). Results obtained with the Bgh isolate K1 (AvrMla1) are shown by closed

columns, and results obtained with isolate A6 (AvrMla6 and AvrMla12) are shown by open columns in

downward orientation. The data shown are means of at least three independent experiments (SD

indicated). Each experiment involved light microscopic examination of at least 100 interaction sites

between a single Bgh sporeling and an attacked epidermal cell. (B) The indicated transgenes and an empty vector control were delivered into epidermal cells of cv

Sultan 5 containing Mla12 Mlo Rar1. Experimental conditions and symbols are identical to those in (A). (C) Transgene Mla12 or an empty vector control was delivered into epidermal cells of two susceptible

Mla12 mutant lines (M66 and M22). Transgene Mla6 or Mla12 or an empty vector control also was

delivered into the rar1-2 mutant line M100. Experimental conditions and symbols are identical to those in

(A).


52

resistance is not effective before haustorium development, consistent with the

previous quantitative inspection of single interaction sites in resistant Mla12

wild-type and susceptible mutant leaves (Freialdenhoven et al., 1994).

However, when the putative Mla12 was over-expressed in cv. Sultan 5

usingthe single cell expression assay, the haustorium index was reduced to

~2%, similar to the level conferred by Mla6 (Fig. 3B). Apparently, over-

expression of the candidate Mla12 shifted the resistance response from post-

haustorium growth arrest to an abortion of fungal development before the

penetration of the epidermal cell wall and the formation of haustoria.

3.3.2 Over-expression of Mla12 complements mutant phenotypes but retains Rar1 dependence

To corroborate the function of Mla12, the over-expression construct of

Mla12 was bombarded into epidermal cells of mutant lines M66, M22, and

M100 (the latter contains the severely defective rar1-2 allele) (Shirasu et al.,

1999a) (Fig. 3C). In these experiments, full AvrMla12-dependent resistance

was restored in both M66 and M22 plants, demonstrating that the mutant

phenotypes were complemented by the candidate Mla12. By contrast, neither

over-expression of Mla6 nor of the candidate Mla12 restored full resistance in

the rar1-2 mutant line M100. The Mla12 over-expression phenotype was

affected more strongly than the Mla6 response in the rar1 mutant background.

The data obtained from over-expression of Mla12 in different genetic

backgrounds strongly support the idea that the RGH in cosmid Sp14-4 is a

functional Mla12.

3.4. Sgt1 is required for Mla12 resistance

Double stranded RNA interference (dsRNAi) gene silencing of HvSgt1

in a single-cell expression system compromised Mla6- but not Mla1-mediated

resistance function (Azevedo et al., 2002). This technique was used to test a

potential SGT1 requirement for Mla12-mediated resistance to Bgh. Detached

barley leaves (Rar1 genotype) were co-bombarded with a dsRNAi vector for


53

silencing Sgt1 and a Mla12 over-expression construct. In comparison to a

control with an empty dsRNAi vector I recorded about 10% increase in Bgh

haustorium index (2% and 13% with and without including dsRNAi effector

construct at 96 h after inoculation), indicating that HvSgt1 is required for

Mla12-triggered resistance.

3.5. Context-dependent function of conserved MLA residues Leu631

and Lys916

The amino acid substitutions in the susceptible mla12 mutants M66

(L631R) and M22 (K916M) affect residues that are conserved in MLA1 and

MLA6, whereas the substitution in mutant M86 (E866K) changes a

nonconserved residue (Figure 1). To investigate the importance of Leu631 and

Lys916 in Mla1- and Mla6-triggered resistance, the same amino acid

substitutions were introduced into Mla1 and Mla6 under the control of the

ubiquitin promoter and were reintroduced into Mla12 for comparison and

confirmation. Wild type and mutant variant plants were tested in the transient

gene expression system. This analysis showed that Mla12 mutant variant

L631R impaired AvrMla12-dependent resistance fully (84%) and K916M

impaired it partially (31%), indicating that the MLA12 (K916M) variant protein

retains residual activity (Fig. 4). This observation is consistent with the fully

compromised and partially impaired Mla12 resistance reported for M66 and

M22 mutant plants (infection types 4 and 2/3, respectively) (Torp and

Jørgensen, 1986) and validates the usefulness of the single-cell assay to

evaluate Mla12 activity using the strong ubiquitin promoter. Surprisingly,

despite an overall sequence relatedness of 90% between the tested MLA

proteins, none of the amino acid replacements in MLA6 or MLA1 resulted in a

detectable change of resistance activity compared with that in the respective

wild-type genes (Fig. 4). Thus, it is possible that other regions are critical for R

protein function in MLA1 and MLA6 (see below). Alternatively, other residues

that are absent or polymorphic in MLA12 might compensate for the functional

contributions of Leu631 and Lys916 in the MLA1/MLA6 substitution mutants.


54

( )m lo-3 ,R ar1

M la1 M la6 M la1 2(L63 1R ) (L631 R) (L63 1R ) (K9 15M ) (K913M ) (K916 M)

M la1 M la 6 M la12 M la1 M la 6 M la12

( )m lo-3 ,R ar1 ( )m lo-3 ,R ar1

Bgh AvrM la 1 K1 ( )

Bg h AvrM la 6,AvrM la 12 A 6 ( )100

100

80

60

40

20

0

20

40

60

80

Haust

orium i

ndex in

GUS e

xpress

ingepi

derma

l cells

(%)

Tr ansgene (& )M lo

G eneticbac kgr ound Figure 4. Context-dependent functions of conserved MLA residues Leu-631

and Lys-916.

Mean values of single cell resistance/susceptibility (%) are shown at left after delivery

of Mla1, Mla6, or Mla12 into the genetic background of cv Ingrid (mlo-3 Rar1).

Results obtained with L631R variants of Mla1, Mla6, and Mla12 are shown in the

middle. Results obtained with Mla1, Mla6, and Mla12 variants each containing a K to

M substitution at the indicated positions are shown at right. Experimental conditions

and designations are identical to those in Figure 2. GUS, β-glucuronidase.


55

3.6 Discussion: Altering resistance response kinetics by Mla dosage

An intriguing feature of Mla-mediated Bgh immunity is the diversity of

macroscopically and microscopically visible infection phenotypes determined

by different Mla R specificities. A quantitative analysis of single interaction

sites in nearly isogenic lines containing different Mla genes revealed for Mla1

and Mla6 early termination of Bgh growth coincident with haustorium

differentiation (Boyd et al., 1995). Rarely were interaction sites found

permitting the development of elongating secondary hyphae. By contrast,

Mla3 and Mla7 mediated cessation of fungal growth at a later stage of the

infection process, permitting frequently the growth of elongating secondary

hyphae on the leaf surface in addition to haustorium differentiation. These Mla

gene-specific differences correlated with the timing of a cell death response

that was either rapid, involving attacked epidermal cells, or slower, including

epidermal and subtending mesophyll cells (Boyd et al., 1995). Similarly, a late

cell death–associated resistance is characteristic for lines carrying Mla12,

permitting indistinguishable fungal growth for up to 36 h after Bgh spore

inoculation and a high haustorium index of ~60% on both Mla12-resistant and

Mla12-susceptible mutant plants (Freialdenhoven et al., 1994). It is possible

that differences in the timing of Mla resistance responses are the indirect

consequence of different infection stage-specific delivery systems for

particular Bgh AVRMLA effector proteins (e.g., delivery of AVRMLA12 only

after or coincident with haustorium differentiation). Precedence for this idea is

found in the expression of Cladosporium fulvum AVR9, which is induced

strongly upon a switch from surface to intercellular growth of the fungus in

leaves, which may be cued by fungal nitrogen starvation (Van Kan et al.,

1991; Perez-Garcia et al., 2001).

In this study it has been shown that slow Mla12-triggered resistance

was altered dramatically to a rapid response by Mla12 over-expression,

leading to almost complete abortion of Bgh attack before haustorium

differentiation (Fig. 3). The retained Rar1 dependence of the Mla12 over-

expression phenotype corroborates this as an authentic but kinetically altered


56

response. Because the rapid response retained AVRMLA12 dependence, the

Bgh effector protein must be, like AVRMLA1 and AVRMLA6 (Halterman et al.,

2001; Zhou et al., 2001) (Fig. 3), delivered before or during the switch from

surface to invasive pathogen growth. The rapid Mla12 over-expression

response suggests that normal cellular amounts of MLA12 or protein

complexes containing MLA12 are rate limiting for the onset or speed of the

resistance. This finding is consistent with previous results demonstrating

markedly reduced resistance in plants that are heterozygous for Mla12 (Torp

and Jørgensen, 1986). Further evidence that MLA levels are regulated in

planta has recently been reported by Halterman et al. (2003). Mla6 and Mla13

transcription levels were induced rapidly between 16 to 24 h post inoculation

of a incompatible pathogen. The time points correspond to that when the

fungus made an intimate contact with the host plant, suggesting the

transcripts accumulations are induced upon the pathogen recognitions

(Halterman et al., 2003).

57

4. Structure and function analysis of MLA protein by domain swapping: the LRR-CT unit in MLA1 and MLA6 determines recognition specificity

4.1. Introduction

Sequence analysis of isolated Mla alleles indicate a common structure

of the CC-NB-LRR-CT type R proteins, sharing over 90% overall sequence

similarity. The most divergent region is the LRR-CT part (~87% sequence

similarity), consistent with the notion that the LRR domain might mediate

protein-protein interaction, and hence the specificity determinant. Despite the

overall similarity in sequence and in the gene structure, isolated Mla

resistance specificities differ in the requirement for their function the Rar1 and

Sgt1 gene (Jørgensen et al, 1996; Freialdenhoven et al, 1994; Schulze-Lefert

and Vogel, 2000; summarised in Table 2). It was assumed that the existence

of a convergence point in signalling pathways triggered by multiple Mla genes

and suggests a function for Rar1 and Sgt1 downstream from pathogen

recognition, whereas the functional independence on Rar1 of Mla1 and other

Mla specificities points to the existence of more than one race-specific

resistance signalling pathway (Schulze-Lefert and Vogel, 2000). How can

both structural and sequence related NB-LRR-CT proteins at Mla locus

mediate distinctive specificities and feed into separate signalling pathways?

4.2. The CT domain in MLA proteins is also subject to diversifying

selection

Attempts to identify regions determining recognition specificity in R-

genes point to the LRR regions in some successful domain swapping

experiments, as well as region outside the LRR domain, for example, the TIR

Structure and function Analysis

58

Table 2. Rar1/Sgt1 requirement of molecularly isolated Mla alleles

Allele Rar1 requirement Sgt1 requirement

Mla1

Mla6

Mla12

Mla13

no

yes

yes

yes

no

yes

yes

yes

Table 3. Comparison of average polymorphic residues in MLA and L NB-

LRR proteins

Region MLAa Lb

TIR

CC

NB-ARC

LRR

CT

--- 1.5

13.0

31.0

19.0

3.9

---

12.0

42.0

---

a Calculated on the basis of MLA1, MLA6, MLA12 and MLA13 b Calculated on the basis of 11 L proteins (L1, L2, L5, L6, L7, L8, L9, L10, L11, L, LH)


59

domain of the L alleles for flax rust resistances can also determine specificity

(Ellis et al, 1999; Luck et al, 2000). A comparison of average polymorphic

residues for MLA and L proteins in different domains revealed that

comparable polymorphism in corresponding domains (Table 3), implicating

again that the respective LRR region is most relevant in specificity

recognition. Unexpectedly, a relatively high polymorphism is also observed in

the CT region of MLA compared to its CC and NB-ARC regions (Table 3).

Moreover, in the middle of the CT domain a hypervariable region was

identified, which shows an increased ratio of nonsynonymous (ka = 15.4) to

synonymous (ks = 9.6) nucleotide substitutions [based on Mla1, Mla6, Mla12,

and Mla13 sequences (Halterman et al., 2003); significant at P < 0.1%],

indicating the operation of diversifying selection and hence an important role

in function or/specificity.

4.3. Domain swaps between MLA1 and MLA6 reveal determinants for

recognition specificity reside in LRR-CT unit

A series of reciprocal domain swaps between Mla1 and Mla6 were

constructed to identify regions that are critical for MLA function and respective

specificities (Fig. 5). The maize ubiquitin promoter drove the expression of

each chimeric gene, and their function was tested after bombardment into leaf

epidermal cells by spore inoculation with Bgh isolates K1 (AvrMla1) and A6

(AvrMla6) at 15 h after delivery. Recognition specificity and activity of the

chimeras were compared with those of the respective Mla1 and Mla6 wild-type

genes whose expression was driven by either native regulatory 5' sequences

or the strong ubiquitin promoter (Fig. 5A). No significantly different activity was

seen using constructs driven by the native or the strong ubiquitin promoter.

Full AvrMla6-dependent recognition specificity was retained in chimeras

containing the complete MLA1-derived CC-NB domains and in chimeras

containing progressively more MLA1-derived N-terminal LRR repeats

(constructs 16666, 11666, and 11166; Fig. 5B). Activities mediated by

chimeras containing only MLA6-derived LRRs 3 to 11 (11661) or only the


60

1 0 0 1 0 0 1 0 01 0 08 0 6 0 4 0 2 0 0 2 0 4 0 6 0 8 0 8 0 6 0 6 04 0 4 02 0 0 2 0 8 0

H austor ium index in G U S express ing c e lls (% ) H austor ium index in G U S express ing c e lls (% )

A B C

Bgh A6 Bgh K1

Rar1 ra r1 -2

M LA 6M LA 6*

16666116661116611116

M LA 1M LA 1*

61111661116661166661

C C N B LR R C T

66116

11661

Bgh A6 Bgh K1

Figure 5. Domain swaps between MLA1 and MLA6 reveal determinants for

recognition specificity and RAR1 dependence.

(A) Schemes of MLA6 (yellow), MLA1 (green), and 10 chimeras are shown. The

relative positions of the CC, NB, LRR, and CT parts are indicated at top, and

acronyms for each chimera are shown at left. The stars indicate gene expression

driven by native 5' flanking sequences; the strong ubiquitin promoter drove the

expression of all other genes.

(B) All genes shown in (A) were expressed in the Rar1 wild-type background, and

mean values of single cell resistance/susceptibility were scored microscopically upon

challenge inoculation with Bgh isolates A6 or K1. Experimental conditions and

designations are identical to those in Figure 2. GUS, β-glucuronidase.

(C) All genes shown in (A) were expressed in the rar1-2 mutant background, and

mean values of single cell resistance/susceptibility were scored microscopically upon

challenge inoculation with Bgh isolates A6 or K1. Experimental conditions and

designations are identical to those in Figure 2.


61

MLA6-derived C terminus (11116) were inactive or severely impaired,

respectively. These data suggest that MLA6 LRRs 9 to 11 act together with

the cognate C-terminal domain to confer AvrMla6 recognition specificity.

Reciprocal domain swaps showed that AvrMla1-dependent activity was

retained upon replacement of the entire MLA1 CC-NB domain only and upon

additional replacement of LRRs 1 and 2 (constructs 61111 and 66111).

Interestingly, longer substitutions up to LRR 8 rendered the 66611 chimera

fully inactive, although the reciprocal construct 11166 fully retained AvrMla6-

dependent activity. Substitutions containing LRRs 3 to 11 (construct 11661)

also compromised AvrMla1 recognition specificity. Because chimeras

containing only MLA1-derived LRRs 3 to 11 (66116) or only the MLA1-derived

C terminus (66661) were inactive, it is therefore concluded that MLA1-derived

LRRs 3 to 11 together with the cognate C-terminal domain are required for

MLA1 recognition specificity.

4.4. Uncoupling the RAR1 requirement from MLA6 recognition specificity

The sequence differences between Mla1 and Mla6 are unusually small,

however, these differences affect both recognition specificity and the use of

Rar1. As more divergence is observed in the LRR-CT region of both MLA, it is

reasonable to assume that region(s) that affect the Rar1 dependence are

probably also located in the LRR-CT region. To gain more insight in that

direction, the activities of wild-type MLA1 and MLA6, as well as the MLA

chimeras were assessed in the rar1-2 genetic background (Fig. 5C). The rar1-

2 mutation leads to a transcript-splicing defect, and a RAR1 antiserum fails to

detect RAR1 signals on protein gel blots (Azevedo et al., 2002). Delivery of

wild-type MLA1 or MLA6 plasmid DNA in rar1-2 leaf epidermal cells led to fully

retained or partially compromised resistance (4 and 39% haustorium index,

respectively) (Fig. 5C). No significant differences were found between wild-

type constructs driven by the native and strong ubiquitin promoters. Thus,


62

Mla6 function is compromised partially by the rar1-2 mutation compared with

bombardments of the same constructs in the Rar1 background (Fig. 5B,C).

Remarkably, delivery of the three chimeras conferring AvrMla6-

dependent resistance in Rar1 plants (16666, 11666, and 11166) displayed

either full RAR1 dependence (constructs 16666 and 11666, each showing

80% haustorium index) or uncoupled RAR1 dependence from recognition

specificity (construct 11166, showing 10% haustorium index) in the rar1-2

background. Neither of the two chimeras that retained AvrMla1-dependent

resistance activity (61111 and 66111) was impaired functionally upon delivery

in rar1-2 mutant plants. Unless MLA6 accumulation is self-limited, the data

suggest that RAR1 dependence cannot be overcome by Mla6 over-expression

and appears to be modulated by both the CC-NB and LRR regions.

It was observed that an Arabidopsis rar1 mutant line failed to

accumulate a CC-NB-LRR protein, RPM1 (Tornero et al. 2002). Therefore it is

reasonable to also test whether MLA6 becomes unstable in the rar1-2 mutant

background. The activity of MLA6 can be inferred as its stability indirectly in

the single cell assay. At 96 h after delivery, MLA6 remained as active as at 15

h after delivery (39% haustorium index), suggesting that MLA6 level required

for its functionality is still present at 96 h after delivery in rar1-2 plants (see

below for examples of unstable MLA variants 16666 and 11666).

4.5. SGT1 is associated with RAR1 in MLA mediated resistances

Previously it was shown that barley Sgt1 (HvSgt1) is required for Mla6-

but not Mla1-mediated resistance using double-stranded RNA interference

(dsRNAi) gene silencing of HvSgt1 in a single-cell expression system

(Azevedo et al., 2002). It is then prompted to examine in the Rar1 wild-type

background the SGT1 requirement of MLA chimeras that retain MLA6

recognition specificity (constructs 16666, 11666, and 11166 in Fig. 5A) using

the same silencing techniques. In these experiments, Bgh spore inoculations

were performed at 48 or 96 h after particle delivery, and the leaf tissue was

fixed for microscopic analysis 48 h after spore inoculation (Fig. 6).


63

M la6 16 66 6 116 66 1116 6

RNA ivector

RNA iSg t1

Bgh AvrM la6,AvrM la 12 A 6 ( )

48 hr96 hr

n.d.

n .d.

*

*

*

10 0

0

20

40

60

80

RNA ivector

RNA iSg t1

RNA ivector

RNA iSg t1

RNA ivector

RNA iSg t1

Haust

orium i

ndex in

G US e

xpress

ingepi

derma

l cells

(%)

Figure 6. Single cell silencing of Sgt1 by dsRNAi.

Wild-type Mla6 or chimeras retaining AvrMla6-dependent recognition specificity were

coexpressed with a HvSgt1 dsRNAi-silencing plasmid (Azevedo et al., 2002) in the

Rar1 wild-type background using a modified single cell transient gene expression

assay (Azevedo et al., 2002). After delivery of plasmid DNAs into epidermal cells,

detached barley leaves were incubated for 48 h (open bars) or 96 h (closed bars).

Subsequently, leaves were inoculated with spores of Bgh isolate A6 (AvrMla6) and

incubated for another 48 h. Microscopic scoring of single interaction sites was

identical to that described for Figure 2. Asterisks indicate haustorium indices that are

significantly different (P < 0.05) from bombardments using empty dsRNAi vector

controls. GUS, β-glucuronidase; n.d., not determined.


64

Co-bombardment of SGT1 dsRNAi DNA with a plasmid driving wild-type Mla6

from the ubiquitin promoter resulted in a small but significantly increased

haustorium index (19% at 96 h after delivery) compared with delivery of an

empty dsRNAi vector control (2%). This result is consistent with previous data

(Azevedo et al., 2002). Unexpectedly, the functioning of chimeras 16666 and

11666 was partially impaired at 48 h after delivery in co-bombardment

experiments with the empty vector dsRNAi control. This phenomenon was

time dependent in that the chimeras were almost completely inactive at 96 h

after delivery. This finding may indicate that the two chimeric MLA proteins are

less stable or that fewer or less active recognition complexes are formed

compared with complex formation in the MLA6 wild-type protein.

Nevertheless, at 48 h after delivery, co-bombardment of plasmids 16666 and

11666 with SGT1 dsRNAi DNA significantly enhanced the haustorium index

compared with that in empty vector controls (P < 0.05), indicating at least a

partial requirement of the chimeras for Sgt1. By contrast, the 11166 chimeric

protein retained full activity upon co-bombardment with the empty dsRNAi

plasmid control, and its function remained unaffected by Sgt1 silencing even

at 96 h after delivery (Fig. 6). Unlike wild-type Mla6, AvrMla6-dependent

resistance conferred by the 11166 variant appears to be uncoupled from both

Rar1 and Sgt1 dependence (Fig. 5C and 6).

65

5. RAR1 is not sufficient to increase MLA steady-state

protein levels in Saccharomyces cerevisiae

5.1. Introduction

It was previously reported that in uninfected Arabidopsis plants steady-

state levels of the R protein RPM1 are greatly reduced in a rar1 null mutant

background (Tornero et al., 2003). Recently, a similar behaviour was

observed for MLA proteins in transgenic barley plants in a rar1 mutant

background (Bieri S., MPIZ unpublished data). MLA6 protein level is

significantly decreased in healthy rar1 mutant plants. Surprisingly, MLA1

protein abundance is also reduced in rar1 mutants although Mla1 resistance

was shown to function independently of Rar1 (Jørgensen, 1996; Zhou et al.,

2001). In Rar1 wild-type plants, MLA1 accumulates to four-fold higher levels

than MLA6, whereas in rar1 mutants MLA1 abundance is decreased to similar

levels as MLA6 in a Rar1 wild-type background. Moreover, protein

accumulation of chimera 11166, previously shown to recognize AvrMla6 and

to function independently of Rar1 (Fig.5 and Chapter 4.4), accumulated to

levels that are indistinguishable from MLA1 abundance in a Rar1 wild type

background. Conversely, steady state levels of chimera 11666, also

recognizing AvrMla6, was much lower than wild type MLA6 abundance in

Rar1 wild type plants. The resistance activity of this chimera was fully

inactivated in a rar1 mutant background (Fig.5 and Chapter 4.4). Given the

fact that RPM1 and MLA are NB-LRR R proteins from dicots or monocots

respectively, it is possible that one function of RAR1 involves stabilization

and/or folding of NB-LRR R proteins. It remains unclear whether this is a

direct or indirect RAR1 activity. MLA protein accumulation appears to be

intrinsically sensitive to elevated temperature in planta because protein levels

of both wild-type MLA1 and MLA6 are markedly decreased after the

RAR1 and MLA stability

66

plants are shifted from 17 oC to 37 oC (Mauch S., MPIZ unpublished data).

This phenomenon is not a general feature of plant proteins as Ponceau

staining revealed an unchanged protein pattern before and after the

temperature shift. In addition, proteins like HSP90, RAR1, and SGT1, that

might regulate MLA stability, do not exhibit temperature sensitivity at the

above tested temperature conditions. It remains unclear whether this

temperature sensitive accumulation of MLA proteins is related to the potential

role of RAR1 in NB-LRR stabilization and/or folding.

To further investigate the role of RAR1 in MLA protein stability, yeast

was used for heterologous expression of MLA proteins in the present study.

MLA1 or MLA6 C-terminal HA-tagged variants, as well as two HA-tagged

chimeric protein variants (11166 and 11666) were expressed in the presence

or absence of HvRAR1 in yeast cells. The potential role of RAR1 in MLA

stabilization/folding was tested at standard yeast growth temperature of 30 oC

and at an elevated temperature of 37 oC. Protein steady-state levels of

respective MLA variants were evaluated and compared by Western blot

analysis of yeast crude protein extracts.

5.2. RAR1 does not alter MLA steady-state protein levels in yeast at standard growth temperature

To express MLAs in yeast, the full-length cDNA of Mla1 and Mla6 were

isolated by RT-PCR and subcloned into a shuttle expression vector under the

control of the inducible GAL promoter. Sequences corresponding to the full-

length cDNA of MLA chimeras 11166 and 11666 were also generated and

subcloned into the same vector. A single HA epitope tag was added to the

end of all Mla cDNA sequences for subsequent immunodetection. Four Mla

containing plasmids were transformed into the same yeast strain using the

LiAc transformation method (Gietz and Woods, 2002) and yeast

transformants were selected by Tryptophan prototrophy.

For RAR1 expression in yeast, the full-length Rar1 cDNA sequence

was fused to a single myc epitope tag at the 3’-end of the sequence and


67

subcloned into expression vector pLexA. This vector served originally as bait

vector in the LexA system and was modified in the present study such that

Rar1 expression is driven by the strong constitutive Adh promoter. In addition,

sequences encoding the LexA DNA binding domain were removed. Presence

of resulting expression vector pQSHvRar1-myc in yeast can be selected by

histidine prototrophy. To achieve co-expression of RAR1 with the tested MLA

derivatives, both Rar1 and respective Mla containing plasmids were co-

transformed into the same yeast strain and colonies were selected for the

presence of both plasmids.

Yeast cultures were raised from strains containing one plasmid to

express individual MLA derivatives alone or in the presence of HvRAR1. The

yeast cultures were grown at 30 oC overnight and subjected to induction for 4

h. Cell pellets were obtained from yeast cultures grown to similar OD600 units.

Crude extracts were made from these pellets by freezing in liquid N2 and then

boiling for 5 min for at least three rounds. Equal volumes of crude extracts

dissolved in loading buffer were separated by SDS-PAGE. After immuno-

blotting with the anti-HA antibody, MLA proteins were detected at the

expected molecular weight in all samples (lane 2 to 9, top panel, Fig. 7).

MLA1-HA expressed in transgenic barley plants showed an electrophoresis

mobility that was indistinguishable from MLA1-HA expressed in yeast (lane1,

Fig. 7). This indicates absence of plant- or yeast-specific modifications of the

MLA protein. RAR1 appears to accumulate to similar levels in all yeast cell

cultures expressing the tested MLA derivatives (bottom panel, lane 6 to 9, Fig.

7). A similar RAR1 level is observed in yeast cells expressing RAR1 alone or

together with MLA proteins (lane 10, Fig. 7). MLA abundance was

indistinguishable for each tested MLA derivative in the presence or absence

of HvRAR1(top panel, lane 2 to 9, Fig. 7). In summary, full-length MLA

proteins can be expressed in the heterologous yeast system but co-

expression of RAR1 is apparently insufficient to elevate MLA steady-state

levels. These finding shows that RAR1 does not directly affect MLA

abundance in yeast or that MLA folding/stabilization might require additional

plant-derived proteins.


68

Plant

Mla1

Rar1-myc

Mla6 1116

611

666

Mla1 Mla6 1116

611

666

25kD

108kDα-HA

α-myc

Mla1

Yeast

empty

Figure 7. RAR1 is not sufficient to alter MLA steady-state protein levels in

yeast.

Individual MLA-HA was expressed in yeast in the absence or presence of RAR1-

myc. Yeast crude extracts were prepared after 6 hr of induction with galactose. Equal

amounts of extract were separated on SDS-PAGE and were immunoblotted with anti-

HA (top pane) or anti-myc (bottom pane) antibody. Crude extracts were from Mla1-

HA transgenic barley leaves (lane 1); or from yeast expressing respective MLA-HA

alone (lane 2-5), or co-expressing with RAR1-myc (lane 6-9); or from yeast

expressing RAR1-myc alone (lane 10).


69

5.3. RAR1 does not impair MLA protein abundance in yeast at elevated temperature

To better understand the role of RAR1 in MLA accumulation, yeast

strains transformed with plasmid expressing various MLA HA-tagged variants

were subject to temperature shift experiments. Yeast cells were tested in the

absence or presence of co-expressed HvRAR1. Cell cultures were cultivated

at 30 oC overnight before induction of MLA-HA expression by galactose.

Individual yeast cell cultures were then divided into two portions; one half was

grown at 30 oC for 2 h and crude extracts prepared from these samples were

used as controls. The other half was shifted to 37 oC for 2 h. All MLA

derivatives accumulated to similar levels at 30 oC in a RAR1 independent

manner (compare lane 2 to 5 with lane 11 to 14, Fig. 8). Likewise, at an

elevated temperature of 37 oC, RAR1 did not significantly alter the abundance

of any of the tested MLA derivatives (compare lane 11 to 14 with lane 15 to

18, Fig. 8). Thus, the observed temperature sensitive MLA accumulation in

planta is not recapitulated in yeast cells despite co-expression of HvRAR1.

This suggests a contribution of additional plant-specific components mediating

in planta temperature sensitivity.

5.4. Discussion

Although biochemical analysis in both Arabidopsis and barley suggests

that RAR1 may have a role in stabilizing NB-LRR R proteins in planta

(Tornero et al., 2002; Bieri et al., unpublished), the exact molecular

mechanism is still unclear. More recently, the cytosolic HSP90 family has

been genetically identified as a component required for the function of some

NB-LRR R proteins, e.g., Arabidopsis RPM1 and RPS2, potato RX, tobacco N

(Hubert et al., 2003; Takahashi et al., 2003; Lu et al., 2003; Liu et al., 2004).

Furthermore, biochemical evidences indicate HSP90 physically associates

with RPM1 or N proteins in planta (Hubert et al., 2003; Liu et al., 2004). It was

also demonstrated that HSP90 is critical for RPM1 and RX stability in plants.

Mutant alleles of one of four genes encoding cytosolic HSP90 severely

compromise RPM1 protein accumulation in Arabidopsis while VIGS silencing


70

α-HA

α-myc 25 kD

108kD

Plant

Mla1

Rar1-mycMla611

166

1166

6

Mla1Yeast

Mla1 Mla61116

611

666

Plant

Mla1

Rar1-myc

Mla61116

611

666

Mla1

Yeast

Mla1 Mla61116

611

666

30 0C 37 0C

Figure 8. RAR1 does not alter MLA steady-state protein levels in yeast at

standard and elevated growth temperature.

Yeast expressing individual MLA-HA alone or co-expressing with RAR1-myc were

grown at standard (30 oC) or elevated (37 oC) growth temperature. Crude extracts

were made from these yeast after galactose induction of MLA expression. Equal

amounts of crude extracts were separated on SDS-PAGE and analysed by

immunoblotting with anti-HA (top panel) or anti-myc (bottom panel) antibodies. Crude

extracts were from Mla1-HA transgenic barley leaves (lane 1 and lane 10); or from

yeast expressing respective MLA-HA alone (lane 2-5 and lane 11-14), or co-

expressing with RAR1-myc (lane 6-9 and lane 15-18).


71

of NbHSP90 impairs RX protein accumulation in Nicotiana benthamiana

(Hubert et al., 2003; Lu et al., 2003). Interestingly, HSP90 has been co-

immunoprecipitated with RAR1 in Arabidopsis, or shown to directly interact

with RAR1 in yeast two-hybrid assays (Hubert et al., 2003; Takahashi et al.,

2003). These findings may indicate that RAR1 assembles together with

HSP90 and R proteins in a hetero-complex. Animal homologs of RAR1 and

SGT1 share structural features (the CS domain) with other co-chaperones

previously shown to bind HSP90 (Dubacq et al., 2002; Garcia-Ranea et al.,

2002). Therefore, it has been proposed that R proteins might be HSP90 client

proteins and RAR1 could function as HSP90 co-chaperone possibly

regulating the stability and activity of NB-LRR R proteins (Hubert et al., 2003;

Takahashi et al, 2003). However, how these heterocomplexes could regulate

R protein activity/stability is still unknown.

In the present study, co-expression of HvRAR1 together with various

MLA derivatives in yeast was not sufficient to alter MLA abundance. These

findings differ from greatly reduced MLA levels seen in planta in rar1 mutant

plants. One possibility is that additional (yet unknown) plant components must

be co-expressed in yeast to mimic the effects seen in planta. Potential

candidates might be HvHSP90 and/or HvSGT1. In this model, RAR1 does not

directly stabilize MLA. It is conceivable that RAR1 stabilizes MLA only if the

latter is physically associated together with other (unknown) host factors in a

presumptive ‘recognition complex’.

MLA protein levels are markedly decreased at elevated temperature in

planta (Mauch S., MPIZ unpublished). This might indicate that MLA proteins

are intrinsically temperature sensitive. Alternatively, presumptive MLA

containing hetero-complexes may become unstable at elevated temperature,

thereby indirectly triggering degradation of free MLA in planta. In yeast cells,

MLA abundance remained unchanged upon a temperature shift from 30 oC to

37 oC, suggesting that MLA proteins are not per se unstable in this eukaryote.

However, since the standard growth condition of barley seedlings was at


72

approximately 20oC, MLA accumulation in yeast should be re-evaluated at a

comparable temperature.

73

6. Identification of MLA interactors using yeast two-hybrid selection

6.1. The LexA yeast two-hybrid system and interaction mating method

6.6.1. The LexA yeast two-hybrid system

The LexA two-hybrid system is a LexA-based interaction trap for

detecting specific protein-protein interactions in yeast (Gyuris et al., 1993;

Mendelsohn & Brent, 1994). In this system the DNA binding domain (BD) in

the bait vector is the entire bacterial LexA protein (Ebina et al., 1983). The

activation domain (AD) in the prey vector is provided by an 88-residue acidic

E. coli peptide (B42) that activates transcription in yeast (Ma & Ptashne,

1987). An interaction between a protein of interest (fused to the BD) and a

library-encoded protein (fused to the AD) creates a novel transcriptional

activator with binding affinity for LexA operators. This factor then activates

reporter genes having upstream LexA operators, which makes the protein-

protein interaction phenotypically detectable. A dual reporter system is used

for detecting protein-protein interactions, consisting of: (i) the LEU2 nutritional

reporter gene, preceded by six copies of the LexA operator to which the DBD

binds, is stably integrated into the yeast genome and its activation allows for

Leucine autotrophic growth selection; (ii) the LacZ gene on an autonomously

replicating plasmid is preceded by eight copies of LexA operator and allows

for a β-galactosidase assay with X-Gal (5-bromo-4-chloro-3-indolyl-β-D-

galactopyranoside) as substrate.

The LexA system provides several advantages compared to other

genetic selection systems for protein-protein interactions. Firstly, the

promoters of the two reporters differ in the sequences flanking the LexA

operators, and this sequence dissimilarity helps to eliminate some false

positive clones and to confirm the positive two-hybrid interactions. Secondly,

the inducible yeast GAL1 promoter drives the expression of AD-fusion

Identification of MLA interactors

74

proteins. Inducible expression provides more opportunity for the prey fusion

proteins with toxicity to the yeast host to survive and thus will less likely be

eliminated from the pool of potentially interacting proteins. Thirdly, both the

LacZ and LEU2 reporters are under control of multiple LexA operators. This

allows several BD-bait fusion proteins to bind to each promoter, thereby

effectively amplifying the intensity of weak signals (Golemis et al., 1996).

6.1.2. The interaction mating method

The interaction mating method takes advantage of the fact that yeast

haploids of two opposite mating type can mate to form diploid cells and hence

presents a convenient method of introducing two different plasmids into the

same yeast cells (Finley & Brent, 1994; Harper et al., 1993). This method

provides at least three benefits:

1). It significantly reduces the labor and time involved in performing a

two-hybrid library screening when several baits will be used to screen a single

library. In this case, different baits transformed into yeast of one mating type

can be used in parallel to screen a library transformed into an opposite mating

type yeast in a single high-efficiency transformation, hence eliminates the

need for many library-scale yeast transformations (which can vary 10- to 100-

fold in their efficiency). It is especially useful when a constitutively expressed

bait interferes with yeast viability. In such cases it is difficult to achieve high-

efficiency transformation.

2). In the interaction mating method the actual selection for interactors

will be conducted in diploid yeast cells, which are more vigorous than haploid

yeast and generally can better tolerate expression of toxic proteins. Thus, it

may improve the chances of finding rare protein-protein interactions.

3). In diploids the reporters are less sensitive to transcription activation

than they are in haploids. It may provide an additional way to reduce the

background from baits that weakly auto-activate transcription of reporters.


75

6.2. Construction of multiple LexA-MLA fusion baits using domains or

full length sequences of MLA1 and MLA6

MLA proteins belong to the CC-NB-LRR-CT subclass of R proteins.

Operationally defined domains or regions in R proteins, e.g. CC, NB-ARC and

LRR-CT, are structurally distinct and are believed to have a distinctive

function (Ellis et al, 2000; Dangl and Jones, 2001; the present study).

Nevertheless, indirect evidence suggests conformational alterations in the

potato RX protein after perception of the corresponding effector protein

(Moffett et al., 2002; Hwang and Williamson, 2003). This suggests that the

full-length protein in non-challenged plants might exist in a closed

conformation that might prevent interactions with a subset of potential host

interactors. To bypass this potential complication, LexA fusion baits were

constructed by using partial cDNA sequences encoding distinct or overlapping

domains (i.e. CC, NB, CC-NB and LRR-CT) of either MLA1 or MLA6. In

addition, fusion baits using both full-length Mla1 and Mla6 cDNA were also

constructed (Fig. 9).

Other considerations were also taken into account when making baits.

First, preliminary observations indicated that fusion bait with the entire MLA1

or MLA6 CC domain activates reporters in the absence of a fusion prey

protein. Therefore, truncations at either C- or N-terminal, or at both ends of

the CC domains were introduced to make multiple CC-containing bait variants

(Fig. 9, only one truncated variant is shown). Second, mutagenesis of genes

encoding the NB-LRR R proteins RX or L identified in either protein single

amino acid substitutions in a conserved motif of the NB-ARC domain that

confers either AVR-effector independent HR, dwarfism, or lethality

(Bendahmane et al., 2002, Jeff Ellis unpublished data). The corresponding

conserved residues in MLA proteins are located at the C-terminal end of the

NB-ARC domain and define a so-called ‘VHDM’ motif . Mutants carrying these

amino acid substitutions confer (Bendahmane et al., 2002, Jeff Ellis

unpublished data). It is proposed that these gain-of-function phenotypes might

disrupt intramolecular interactions in the NB-ARC region (Bendahmane et al.,


76

MLA6

MLA1

CC NB-ARC LRR CT

**

CC (1-166)

CC-NB(D502V)

CC-NB(H501A)

NB

CC-NB

CC (1-166)

Mla-CC∆ (1-43)

NB

CC-NB

LRR-CT

LRR-CT

CDS

CDS

MLA (108KDa)

Figure 9. Graphic representation of MLA full-length protein and MLA baits

used for yeast two-hybrid screenings.

Modular structure of MLA are shown on the top. The name of the baits are shown on

the right. The Mla-CC∆(1-46) bait containing a truncated CC domain and is

conserved between MLA1 and MLA6 sequence. The relative position of the amino

acid substitution in the VHDM motif is shown in star in two mutated versions of

MLA1-CC-NB bait.


77

2002). To increase the chances of identifying MLA interactors, single amino

acid substitutions were introduced into the VHDM motif in MLA1, generating

fusion baits MLA1-CC-NB (H501A) or MLA1-CC-NB (D502V). In total, 18

different LexA-MLA fusion baits were constructed (Fig. 9, some autoactivating

baits are not shown).

6.3. Transforming yeast strain EGY48 (MATα) with bait plasmid and

characterization of bait strains

To perform a yeast two-hybrid screen by the interaction mating method,

the bait plasmids and the prey library DNA were separately pre-transformed

into two yeast strains of different mating types. The resulting bait strains were

then tested for reporter autoactivation and only those that did not activate the

reporters were used for subsequent screenings. Individual MLA bait

constructs were transformed into the yeast strain EGY48 (mating type: MATα)

that carries an autonomous plasmid (p8op-LacZ) in which the LacZ reporter

gene is integrated. All resulting bait strains were then tested for reporter gene

activation by each baits itself. Unexpectedly, almost all baits containing the

CC domain autoactivated both reporters (LacZ and the LEU) for unknown

reasons, except MLA-CC∆(1-46) that contains only the first 46 amino acids of

the MLA N-terminus. Other baits did not activate the two tested reporters.

Notably, baits containing either wild type MLA1-CC-NB or MLA6-CC-NB were

not autoactive, and the same is true for two variants containing mutant VHDM

versions, MLA1-CC-NB(H501A) and MLA1-CC-NB(D502V), although they

contain the intact CC domain.

Some bait strains were also tested for the expression of the fusion

proteins. Crude protein extracts from individual bait strain cultures were

prepared and separated by SDS-PAGE. The fusion proteins were detected by

immuno-blotting using a commercial LexA-antiserum. All bait strains tested

express individual bait proteins to a level that was easily detectable (Fig. 10).

However, two full-length fusions (MLA1-CDS and MLA6-CDS) proteins

accumulated to lower levels compared to other bait proteins (Lane 5 and 11,


78

M l a 1 - CC

M l a 1 - NB

M l a 6 - NB

Mla1-LRR-CT

Ml a1 -CDS

L e x AM l a 6 - C

C

Ml a1-CC-N

B

Mla6-CC-N

B

Mla6-LRR-CT

M l a6 -CDS

96K D

52K D

36K D

29K D

α -LexA

Figure 10. LexA-MLA fusion bait proteins are expressed in yeast.

Yeast culture of respective bait yeast strains were raised overnight. Crude extracts

were made from these cultures and separated on SDS-PAGE. Anti-LexA antibody

was used for subsequent immunoblotting detection of bait protein expression. Crude

extracts were from yeast expressing either full-length CC domains (lane 1 and 7),

NB-ARC domains (lane 2 and 3), LRR-CT regions (lane 4 and 10), CC-NB-ARC

regions (lane 8 and 9), both full-length proteins (lane 5 and 11), or the LexA DNA

binding domain alone (lane 6).


79

Fig. 10). In addition, the size of these two fusion proteins is smaller than the

expected full-length protein (~130 KDa), indicating a possible truncation from

the C-terminal ends for unknown reasons (Lane 5 and 11, Fig. 10).

6.4. A barley prey library suitable for yeast two-hybrid selection by

mating type

A barley prey library was created using cDNA synthesized from

poly(A)+RNA isolated from mixed leaf tissue samples of barley cultivar

Sultan5. Samples were collected from healthy leaf tissue and from Bgh

challenged leaf material at 6, 12, and 24 h post inoculation with spores of an

incompatible A6 isolate (Piffanelli P., and Schulze-Lefert, unpublished data).

High purity library plasmid DNA was obtained from crude DNA preparations

from E. coli cell cultures using the CsCl gradient ultra-centrifugation method.

Subsequently, the library DNA was used to transform the YM4271 strain

(mating type: MATa). In total, ~ 2 x 106 independent yeast transformants were

obtained. This prey library was amplified when the transformants were

selected and collected from selection media. Glycerol stocks of this library are

kept as individual aliquots at -80 oC and can be used for multiple mating type

protein-protein interaction screens. The plating efficiency of the frozen cells

was determined thereafter at about 5x107 CFU/100µl for library glycerol

stocks.

6.5. Library screenings using interaction mating methods

For interaction mating, the haploid yeast strain containing the MLA bait

was mated with the haploid yeast cells expressing individual prey to allow

formation of diploid yeast cells. Briefly, respective EGY48 (p8op-LacZ)

(MATα) bait yeast cells were used to raise overnight cultures and appropriate

OD units of these cultures were combined with YM4271(MATa) cells (ratio of

2.5:1).. The mixtures were subjected to mating procedures and the resulting

diploid cells were then induced for the expression of the AD-library proteins.

On average, mating efficiency was determined to be ~10% (the ratio of

zygotes versus viable MATa cells). On average, each of the baits screened


80

~10x107 viable library cells and ~10x106 diploid cells (i.e. double

transformants) were obtained from each screening. Approximately 6x106

diploid cells were plated on inducing synthetic dropout plates lacking Leucine

(Gal/Raf/SD/-Leu) to select for positive interaction events between individual

bait and library prey fusion proteins that activate the LEU reporter. The

appearance of colonies was checked during 2-5 days after plating. Individual

colonies emerging on the selection media were picked to proceed for

subsequent analysis (Fig. 11 for an overview).

6.6. Characterization of cDNA clones isolated from the prey library

6.6.1. Eliminating false positive clones

False positive clones are common in yeast two-hybrid screenings. Two

major types of false positive clones might arise in the LexA system. First,

some AD-prey fusions may interact with the LexA operator flanking promoter

sequence, which may activate the reporters. Second, activation of reporter

might occur independent of the expression of a library cDNA prey protein. The

former class of false positive clones can be eliminated by using the second

reporter (LacZ) differing in promoter sequences from the one in the LEU

reporter. The latter type of false positive clones can be eliminated by

assessing reporter gene activation under conditions in which the prey proteins

are not induced. All colonies picked were replicated on non-inducing media

plates to turn off the expression of library proteins under the control of GAL1

promoter in the prey vector. Colonies were replicated to different indicator

plates to reveal these two major types of false positive clones in the LexA

system according to the above criteria.

By comparison of the colony numbers before and after the secondary

selection described above, it became obvious that most MLA baits identified

many false positive interactors from the library (compare lane 2 and 3, table

4). Only few MLA baits identified most of the potential interactors (lane 3,

table 4). For example, screenings using MLA-CC∆(1-46) or MLA1-LRR-CT as

bait identified 154 and 72 clones after false-positive elimination steps,


81

Frozen library aliquots

Figure 11. Diagram of major steps of a yeast two-hybrid screening using the interaction mating method. Expression of the library fusion protein is induced in galactose and raffinose containing medium. Further characterizations of putative interactors are not shown here.

EGY48MATa

Selection: Gal/Raf/SD/-UHTL

TestingMating

efficiency

Mating and inducing library protein expression

YM4271MATα

EGY48(p8op-lacZ,MATa)DBD/ bait fusion

YM4271(MATα)AD/library fusion

Report or Baits transformation

Librarytransformation

Auto-activation testing

Baitstrain

Preystrain

Expected result if two hybrid proteins interact

Expected result if two hybrid proteins do not interact

(a) SD/-UHT+ X-Gal + BU

(b) Gal/Raf/SD/-UHT+ X-Gal + BU

(c) SD/-UHTL

(d) Gal/Raf/SD/-UHTL

Re-plating

Baitinsert

Libraryinsert

1 2

Turn off pGAL1: SD/-UHT

1 2


82

respectively. To process the large number of remaining prey clones, it was

necessary to group these further using a DNA fingerprinting method.

6.6.2. Discriminating non-redundant clones from redundant ones

It was expected that several identical prey clones would have passed

the genetic selection if the corresponding mRNA accumulates constitutively to

high levels or increases in abundance upon Bgh pathogen challenge. To

eliminate such redundant clones, cDNA inserts were PCR amplified from each

prey plasmid DNA using primers flanking the inserts. Amplified products were

subsequently digested with restriction enzymes that recognize frequently

occurring restriction sites in cDNAs (e.g. HaeIII). Those prey clones

displaying identical digestion patterns after gel electrophoresis likely represent

either identical or highly sequence-related cDNAs. By this means it was

possible to identify 89 distinct prey cDNAs with an average insert size of

1.2 kb. Thus, the molecular fingerprinting method eliminated about 90% of the

genetically selected 824 prey clones. DNA sequencing was then used for

further classification of the identified 89 prey cDNA clones (table 4).

6.6.3. MLA proteins/domains associate with structurally distinct host proteins

A potential sequence relatedness of the identified MLA interactors with

known proteins was analyzed using either direct nucleotide-nucleotide BLAST

(blastn) or translated polypeptide protein-protein BLAST (blastp) algorithms

(Table 5). Some deduced MLA interactors were identified several times in the

yeast two-hybrid screens described above although the molecular

fingerprinting of the corresponding cDNA clones indicated in many cases

clear differences between individual clones. For example, oxygen evolving


83

Table 4. Selection of prey clones interacting with MLA baits during yeast

two-hybrid screenings by interaction mating methods

BD bait

Colonies picked

from selection

media

Colonies after

elimination of false

positive clones

Prey clones used

for sequencing

after DNA

fingerprinting

Mla-CC∆(1-46) 188 154 38

Mla1-CC-NB 20 0 0

Mla6-CC-NB 24 1 1

Mla1-CC-NB(H501A) 50 4 4

Mla1-CC-NB(D502V) 94 8 4

Mla1-NB 94 3 3

Mla6-NB 24 1 1

Mla1-LRR-CT 116 72 32

Mla6-LRR-CT 94 1 1

Mla1-CDS 72 0 0

Mla6-CDS 48 5 5

In total 824 249 89


84

20

10

2

1

1

1

29

1

Oxygen evolving protein

Carbonic anhydrase

Putative WRKY TF

bZIP TF

Zn transporter

Formin protein

SGT1

Unknown protein

3-isopropylmalate dehydrogenase

Bait

Times identified aMLA interactors

Mla6-LRR-CT

Mla1-LRR-CT

Mla6-CDS

Mla6-NB

Mla1-NB

Mla1-CC-N

B(D502V)

Mla1-CC-N

B(H501A)

Mla-CC

∆ (1-46)

+

+

+

+

+

+

+

+

+ + + + + +16

a The same interactor may be isolated independently several times as indicated

because cDNA clones of the same interactor of variable length are present in the

prey library.

+ denotes interaction.

Table 5. Deduced functions of MLA interactors isolated by yeast two-

hybrid screenings.


85

protein and carbonic anhydrase were identified by the MLA-CC∆(1-46) bait 20

and 10 times, respectively (Fig. 12; Fig. 13). Length variations of cDNAs

encoding oxygen evolving protein and carbonic anhydrase can explain why

many prey clones were originally assigned to different groups after the DNA

fingerprinting. Because DNA sequences were obtained only from the coding

DNA strand, comprising on average about 600 bp of prey cDNA, the

sequences might still contain few sequencing errors. Despite this caveat, the

alignment of carbonic anhydrase cDNA sequences obtained from 10

independently isolated prey clones provides no evidence that these are

derived from different sequence-diverged gene family members (see

Discussion below). Likewise, the 20 prey cDNA sequences encoding variable-

length oxygen evolving proteins are also likely derived from the same gene.

Two prey clones each encoding a putative WRKY transcription factor

were also isolated by the MLA-CC∆(1-46) bait (Table 5). Finally, single prey

clones encoding either proteins with homology to bZIP type transcription

factors, or Zn2+ transporters, or a Formin related protein associated with the

MLA-CC∆(1-46) bait. Interestingly, HvSGT1 was isolated from the library 29

times by the MLA1-LRR-CT bait but not by the corresponding MLA6-LRR-CT

bait. This is remarkable since these two MLA LRR-CT regions share ~87%

amino acid sequence identity (Table 5).

Six different MLA1 and MLA6 baits identified prey encoding 3-

isopropylmalate dehydrogenase (Leu2 in yeast nomenclature). This enzyme

convert β-isopropylmalate to α-ketoisocaproate in the Leucine biosynthesis

pathway operated in eubacteria, archaebacteria, fungi and plants (Kohllaw

2003). This pathway is believed to be of ancient original and the catalytic

functions of the enzymes in this pathway are largely unchanged throughout

evolution (Kohllaw 2003). In the present yeast two-hybrid screening, one of

the selection markers used is Leucine prototrophy. It is possible that the prey

clones encoded 3-isopropylmalate dehydrogenase complemented the leu2

auxotrophic mutant phenotype and suvived the selection. Thus, these prey

clones are most likely false positives. Indirect evidence comes from DNA

sequence similarity analysis, which showed that these prey cDNA sequences


86

Putative Hv oxygen evolving protein (33KDa)

2

2

3

4

1

4

1

1

2

No. of prey clones identified

MSP

Figure 12. Graphic representation of putative barley oxygen evolving protein and the prey

clones identified in the screening by the MLA-CC∆(1-46) bait.

The relative start and end position of the prey clones are deduced by sequence

comparison to the putative barley oxygen evolving protein. The same prey clone was

identified independently different times as indicated. MSP, Manganese-stabilising

protein conserved domain.


87

HvCA(35KDa)

2

8


Figure 13. Graphic representation of barley carbonic anhydrase and deduced

prey proteins identified in the screening by the MLA-CC∆(1-46) bait.

The relative start and end position of the prey clones are deduced by sequence

comparison to the barley carbonic anhydrase. The same prey clone was identified

independently different times as indicated.


88

encoding 3-isopropylmalate dehydrogenase share higher similarity to fungus-

derived 3-isopropylmalate dehydrogenase cDNAs compared to the homologs

present in the barley EST database. It is noteworthy that the leaf samples

used for prey library construction were taken at various time points after Bgh

spore inoculations. It was expected that mRNA made from these samples

contains also Bgh-derived cDNAs. Therefore, it is likely these cDNA inserts

are derived from the Bgh pathogen.

6.7. Summary and perspective

A yeast two-hybrid screening method that exploits mating of yeast

strains with opposite mating types was used to identify MLA interactors. A

major advantage of this approach is the possibility of using frozen aliquots of

an established prey library stock in yeast strain of one mating type; hence

transformation of the prey library in every single screening becomes

unnecessary. In the present study, efforts have been made to combine the

interaction mating method with the LexA-based yeast two-hybrid system.

Extensive optimizations have been applied to mating, selection and

subsequent clone characterization steps. A barley cDNA prey library was

transformed into yeast strain YM4271 (mating type:MATa). After amplification,

aliquots of this library were kept as yeast glycerol stocks. It was demonstrated

here that it is possible to screen this library many times independently, each

time with several baits in parallel. This substantially reduces the time and the

costs normally associated with conventional selection methods, enabling

parallel screens with diverse baits (Soellick and Uhrig, 2001).

A total of 18 different baits, encoding single domains, two domains or

full-length MLA proteins, were originally constructed for the interaction mating

screenings. Upon elimination of auto-active baits, 11 could be used to perform

screenings. Two baits, MLA-CC∆(1-46) and MLA1-LRR-CT, identified

potentially interesting interactors. One possibility is that these MLA domains

preferentially mediate protein-protein interactions. It is worth noting that most

of the N-terminal MLA baits containing a full-length CC domain auto-activated

the reporters whereas both tested wild type CC-NB-ARC baits did not. This


89

may suggest that in the CC-NB-ARC baits the CC domain interacts

intramolecularly with the NB-ARC region, thereby forming a closed

conformation that prevents interaction with prey proteins. This is consistent

with experimental data indicating that RX protein activation involves

disruptions of intramolecular domain interactions, including the CC and NB-

ARC (Moffett et al., 2002). Furthermore, the MLA1-CC-NB(H501A) and

MLA1-CC-NB(D502V) did not identify any interacting proteins. This might

indicate that the amino acids substitutions introduced in the VHDM motif of

MLA are not sufficient to disrupt the potential intramolecular interaction

between the CC and NB-ARC domains.

HvSGT1 was identified several times by the MLA1-LRR-CT bait (Table

5; Fig. 14). Since SGT1 has been shown to be required for many R gene

mediated resistance responses, the detected physical association in yeast

may have significance in planta. Interestingly, the finding reported here is

reminiscent of the physical association between yeast SGT1 and the yeast

LRR-containing adenylyl cyclase (Dubacq et al, 2002). Recently it has been

demonstrated that Arabidopsis or barley SGT1 co-immunoprecipitates with

HSP90 in planta (Hubert et al., 2003; Takahashi et al., 2003). Furthermore,

SGT1 shares structural features (the TPR and CS domain) with Hop/Sti1 and

p23 co-chaperones (Dubacq et al., 2002; Garcia-Ranea et al., 2002). It has

therefore been proposed that SGT1 might act as co-chaperone in plants to

bind to HSP90 and to regulate R protein stability/activity (Hubert et al., 2003;

Lu et al., 2003; Takahashi et al., 2003; Liu et al., 2004). In this context it would

be interesting to find out whether yeast HSP90 is also involved in the MLA1-

LRR-CT and SGT1 interaction. By extrapolation, it is conceivable that SGT1

associates with MLA proteins in a complex containing HSP90. (see also

General Discussion).

Barley carbonic anhydrase (CA) was isolated several times with the

MLA-CC∆(1-46) bait (Table 5). So far two CA isoforms are known to exist in

the barley genome and differ only in the 3’ untranslated region (Bracey and

Bartlett., 1995). The transcripts are derived from nuclear genes but the


90

HvSGT1 (40KDa)

1

4

5

3

16

TPR CS SGS


Figure 14. Graphic representation of HvSGT1 protein and the prey clones

identified in the screening by the MLA1-LRR-CT bait.

The modular structures of the HvSGT1 protein are shown on the top and the

structures of the prey clones were deduced by comparison to the HvSGT1 sequence.

The same prey clone was identified independently different times as indicated.


91

encoded proteins are transported into chloroplasts. It is unknown yet whether

other barley CA isoforms are present in the barley genome. Tobacco CA was

previously shown to bind to salicylic acid (SA) and is thought to contribute to

antioxidant activity by complementing the ability of a CA-like deletion yeast

strain to grow aerobically (Slaymaker et al., 2002). Interestingly, silencing of

CA gene expression in tobacco leaves suppresses the Pto:avrPto-mediated

HR response, indicating that CA is required for a cell death response upon

activation of race-specific resistance. In Mla mediated resistance responses,

HR cell death is normally confined to epidermal and underlying mesophyll

cells at sites of attempted fungal invasion (Hückelhoven et al., 1999). It would

be interesting to find out whether barley CA is involved in the HR response

that is mediated by MLA R proteins. One might speculate that the biotrophic

Bgh fungus evolved means to manipulate (inactivate) host factors that are

required for R protein triggered resistance responses. In this scenario, MLA

proteins might survey (guard) CA proteins that are in transit to the chloroplast.

In this context, it should be interesting to find out whether Bgh AVR effectors

associate with barley CA.

A barley Formin homolog containing the Formin Homology 2 Domain

(FH2) was also identified by the MLA-CC∆(1-46) bait. Formin proteins in

animals and fungi control rearrangements of the actin cytoskeleton and are

involved in cell polarity processes (Deeks, M., et al., 2002) ). Members of this

family have been found to interact with Rho-GTPases, profilin and other actin-

associated proteins and also play roles in signal transduction processes. Both

pharmacological and genetic evidence suggest that actin cytoskeleton

rearrangements and processes controlling cell polarity are involved in

resistance responses to Bgh at the cell periphery. These broad-spectrum

resistance responses to powdery mildews and other fungal pathogens prevent

a switch from surface to invasive fungal growth (Collins et al., 2003).

AvrMla10 and Avrk1 have been recently cloned from the Bgh genome

(Ridout et al., JIC, unpublished). Sequence comparison of the deduced

proteins encoded by these two Avr genes revealed a conserved core region


92

sharing 56% identical residues whereas both N- and C-termini are highly

sequence divergent. This provides a future opportunity to identify host factors

that might be targeted by the fungal effectors using the yeast two-hybrid

screening system established in this work. Multiple LexA-AVR fusion baits are

under construction and these baits will be used to screen the barley prey

library (see also General Discussion).

93

7. General discussion

7.1. Allelic variants encode MLA R proteins

Eight NB-LRR genes are present in a 260-kb interval comprising the

Mla locus in barley cv Morex and were classified in three dissimilar families

(RGH1, RGH2, and RGH3) with <43% amino acid sequence similarity

between families (Wei et al., 2002). Computational analysis of the Morex 260-

kb sequence contig suggested that a progenitor Mla locus harbored at >8

million years before the present one member of each RGH family (RGH1bcd,

RGH2a, and RGH3a) (Wei et al., 2002). Each of the Mla powdery mildew R

genes identified to date shows highest overall sequence similarity to Morex

RGH1bcd in coding regions and shares the same exon/intron structure (Fig.

15) (Wei et al., 2002). Unlike RGH1bcd, however, each of Mla1/6/12/13

contains a 5' untranslated open reading frame and, within intron 3, an (AT)n

simple sequence repeat consisting of different repeat numbers (Fig. 15). Also,

Morex RGH1bcd contains a BARE1 solo long terminal repeat in intron 3 that is

absent in Mla1/6/12/13, and the presence of a 29-bp deletion in the LRR

region, resulting in a premature stop codon, suggests that it is nonfunctional

(Fig. 15). Because Morex lacks a known Mla powdery mildew resistance

specificity, it has been inferred that RGH1bcd is a naturally inactive allele that

may have served as progenitor for the other Morex RGH1 family members

(RGH1a, RGH1e, and RGH1f) (Wei et al., 2002).

DNA gel blot analysis and preliminary sequence information obtained

from near-isogenic barley lines containing other Mla powdery mildew

resistance specificities indicate for each line the presence of at least one

candidate gene with high sequence relatedness to MLA1/6/12 (Bieri S.,

Pajonk S., et al., MPIZ, unpublished). Thus, it is possible that most if not all

genetically characterized powdery mildew R genes at Mla are alleles of the

General Discussion

94

M la1

M orex m la1 7 8k195 k

1 9k

1 2kM la6

1

1 6k

1

[AT]8

6 2k6 6k

M la12

u O RF

u O RF

u O RF [AT]3 6

[AT]1 4

M la 1

M la 1 2

M la 6

RG H 1b cd

M la 1 -2

RG H 3b

1 0 2k1 06 k RG H 3a

8 1k8 7k RG H 1f

1 2 1k1 27 k RG H 1eor o r

Figure 15. Schemes of the Morex Mla locus and genomic regions containing

identified Mla resistance genes.

DNA sequences encompassing the Morex Mla locus (261 kb, in reverse orientation)

(Wei et al., 2002) are represented schematically and drawn to scale in the top line

(relevant sequences only). Available genomic sequences of Mla1, Mla6, and Mla12

and flanking regions are shown below. Coding sequences of functional Mla R genes

and RGHs are boxed and highlighted in black and blue, respectively. A conserved

upstream open reading frame (uORF) and a simple [AT]n microsatellite are shared

among functional Mla R genes. Green boxes denote retrotransposon sequences: a

BARE1 solo LTR in intron 3 of RGH1bcd, HORPIA2 immediately 3' of RGH1bcd, and

ALEXANDRA 5' of RGH1bcd. Dark gray areas denote sequences showing >90%

identity, and light gray areas denote sequences showing >75% identity. A possible

inversion event could explain the altered relative orientations of homologous genes

Mla1-2 and RGH1f as indicated. Note that RGH1e/f and RGH3a/b are extremely

similar and located within a 40-kb duplicated region (Wei et al., 2002). For this

reason, the indicated homologies exist between RGH1e and RGH1f and between

RGH3a and RGH3b. Arrows indicate the relative orientations of genes (5' to 3').

Borders of Morex sequences are indicated in kb according to accession AF427791.

General Discussion

95

molecularly validated Mla resistance specificities to Bgh. The presence of the

(AT)n microsatellite in all Mla R genes identified to date and its absence in

currently available Mla RGHs are consistent with this hypothesis, because

recent findings indicate that most microsatellites reside in regions predating

recent genome expansion in plants (Morgante et al., 2002).

The very high level of DNA sequence conservation in exon and intron

sequences of identified Mla R genes (average overall identity of 94 and 93%,

respectively) may be indicative of selective constraints acting on both coding

and noncoding regions. By contrast, inspection of flanking regions revealed

evidence for extensive intralocus recombination events that reshuffled both

genes and intergenic regions (Fig. 15). For example, sequences located

immediately 3’ of Mla12 were found 5.5 Kb downstream of RGH1bcd,

indicating an extensive intralocus insertion/deletion event. Morex RGH1f/e

exhibited highest sequence relatedness to the Mla1 paralog Mla1-2; their

altered relative orientation to RGH1bcd and Mla1, respectively, suggests the

occurrence of an intralocus inversion event (Fig. 15).

7.2. Determinants of MLA recognition specificity

Functional analysis of reciprocal domain-swap constructs between Mla1

and Mla6 revealed an essential role of the LRR-CT unit in specificity

determination (Fig. 5B). It was demonstrated in this study that distinct regions

in the LRRs of MLA1 and MLA6 (LRRs 3 to 11 and 9 to 11, respectively) are

necessary for cognate AVRMLA perception. This finding is in agreement with

LRRs representing the most variable part of MLA and other characterized NB-

LRR-type R proteins (Botella et al., 1998; McDowell et al., 1998; Meyers et al.,

1998; Ellis et al., 1999; Halterman et al., 2001). It is also consistent with the

finding that potentially solvent-exposed residues in MLA LRRs and those of

other NB-LRR R proteins are subject to diversifying selection (Botella et al.,

1998; McDowell et al., 1998; Meyers et al., 1998; Halterman et al., 2001). One

interpretation of these data is that the diversified regions are involved in

ligand-specific recognition.

General Discussion

96

LRRs have been demonstrated to function as specificity determinants

of membrane-anchored R proteins (Van der Hoorn et al., 2001; Wulff et al.,

2001). Successful domain-swap experiments have been reported only for

intracellular TIR- NB-LRR-encoding resistance alleles at the L locus in flax to

the flax rust fungus (Ellis et al., 1999; Luck et al., 2000). Both MLA and L

proteins exhibit comparable average polymorphisms in corresponding

domains (Table 3). Unlike this study involving CC-NB-LRR proteins, the

analysis of L chimera functions suggested that both TIR-NB and LRR regions

can determine specificity differences (Ellis et al., 1999; Luck et al., 2000).

Although it is possible that the CC-NB domain is irrelevant for specificity

determination, more diverged CC-NB domains from other MLA proteins must

be tested before any generalization can be made from the observations based

on MLA1 and MLA6 chimeras.

Reciprocal swaps of the CT domains between MLA1 and MLA6

resulted in nonfunctional chimeras (11116 and 66661; Fig. 5B). One

interpretation is that cognate LRR-CT units are required for MLA specificity

determination, which was also supported by the finding that two of three

single-amino acid replacements in mutant MLA12 variants affect CT amino

acids and the third affects an LRR residue (Fig. 2). Additional evidence for a

role of the MLA CT in specificity determination comes from the identification of

a hypervariable region in the middle of this domain (residues 893 to 945 in

MLA1). This hypervariable region shows an increased ratio of

nonsynonymous (ka = 15.4) to synonymous (ks = 9.6) nucleotide substitutions

(based on Mla1, Mla6, Mla12, and Mla13 sequences (significant at P < 0.1%),

which is indicative of the operation of diversifying selection. This is reminiscent

of the C-terminal non-LRR domain of P locus genes that encode flax TIR-NB-

LRR proteins, which also was found to contain a region that is subject to

diversifying selection and might contribute to specificity determination (Dodds

et al., 2001).

7.3. Potential roles of RAR1 and SGT1 in MLA-mediated resistances

General Discussion

97

By genetic studies it has been well documented that RAR1 is required

for members from both TIR- and CC- NB-LRR subtype R proteins against

oomycete, bacterial, fungal and viral pathogens (Freialdenhoven et al, 1994;

Jørgensen, 1996; Liu et al., 2002a; Muskett et al., 2002; Tornero et al., 2002).

Likewise, gene silencing or loss-of-function alleles of sgt1 demonstrated that

SGT1 also plays an important role in race-specific disease resistance to

different pathogen classes mediated by the two major structural subtypes of

NB-LRR R proteins (Azevedo et al., 2002; Austin et al., 2002; Liu et al.,

2002b; Peart et al., 2002; Tör et al., 2002). Significantly, VIGS mediated

silencing of Sgt1 in Nicotiana benthamiana demonstrated that SGT1 is also

required for certain non-host resistance responses (Peart et al., 2002).

Furthermore, SGT1 physically associates with RAR1 in plant and in yeast,

and both proteins co-operate in some R protein mediated resistance against

different pathogens (Azevedo et al, 2002; Austin et al., 2002; Liu et al.,

2002b).

Genetic analysis also provided strong evidence that RAR1 is essential

for the function of a subset of Mla-encoded R specificities (Jørgensen, 1988;

Freialdenhoven et al., 1994). Gene silencing in single barley epidermal cells

of Rar1 and/or Sgt1 significantly compromised Mla6 and Mla12-mediated

resistance. In contrast, Mla1 resistance was only slightly affected in these

experiments (Azevedo et al., 2002; this study). Using transient gene

expression in rar1 mutant plants, Rar1 was shown to be required for Mla13-

mediated resistance (Halterman et al., 2003).

A rapid and coordinate increase in the accumulation of Mla13

transcripts, and Rar1 and Sgt1 transcripts was observed within roughly the

same time frame when Bgh haustoria make contact with the host cell plasma

membrane (around 16-24 h after inoculation; Halterman et al., 2003). It

remains unclear whether the pathogen responsiveness of Mla, Rar1, and Sgt1

gene expression is required for an effective resistance response. In the

present study, functional analysis of chimeras derived from MLA1 and MLA6

shed more light on the role of RAR1 and SGT1 in disease resistance. AvrMla6

General Discussion

98

specificity was successfully uncoupled from the RAR1/SGT1 dependence in

one chimera, suggesting that the reliance on RAR1/SGT1 is not absolute for a

given Mla recognition specificity. One implication from this finding is that

RAR1/SGT1 cannot have a role in processing or transport of Bgh AVR

effectors.

7.3.1. The Ubiquitin/26S proteasome degradation pathway and MLA-

mediated resistance are connected to RAR1/SGT1

The ubiquitin (Ub)/26S proteasome system is a proteolytic pathway

that selectively targets proteins for degradation in eukaryotic cells to regulate

the activity of crucial cellular regulators (Hershko and Ciechanover, 1998;

Callis and Vierstra, 2000; Sullivan et al., 2003). In this pathway, targeted

proteins are marked by ubiquitylation through an ubiquitin conjugation

cascade, which involves three enzyme families, ubiquitin-activating (E1), -

conjugating (E2), and -ligating (E3) enzymes (Glickman and Ciechanover,

2002). Subsequently, the polyubiquitinated proteins are recognized and

degraded by the multi-subunit 26S proteasome that consists of a 20S core

particle flanked by 19S regulatory complexes (Hershko and Ciechanover,

1998; Vierstra, 2003).

One type of complex E3 ubiquitin ligases is the SCF (SKP1,

CDC53p/or Cullin1, F-box protein) complex, which consists of four subunits

(RBX1/or ROC1/or HRT1 being the fourth subunit) (Deshaies, R.J., 1999).

Specificity of the SCF complex is conferred by the F-box subunit that contains

a protein-interaction motif at its C-terminus and an F-box motif at its N-

terminus (Gagne et al., 2002). The F-box protein recognizes and recruits a

specific substrate to the SCF complex for ubiquitylation (Craig and Tyers,

1999). There are also other types of E3 ubiquitin ligases described based on

subunit composition and mechanism of action, for example, Ring/U box,

HECT, VBC-Cul2 and APC (Vierstra, 2003).

Evidence obtained from mutant analysis has already pointed out that

General Discussion

99

the Ubiquitin/26S pathway might be linked to plant disease resistance. For

example, two F-box proteins, CORONATINE INSENSITIVE1 (COI1) and

SUPPRESSOR OF nim1-1 (SON1), have been identified and connected to

disease resistance responses (Xie et al., 1998; Kim and Delaney, 2002). The

Arabidopsis coi1 mutant is unable to express the JA-inducible gene PDF1.2

and is susceptible to insect herbivory and to fungal and bacterial pathogens

(McConn et al., 1997; Thomma et al., 1998). COI1 co-immunoprecipitates

with other SCF subunits (SKP1, CUL1 and RBX1), suggesting a SCFCOI1

complex in planta. This complex is thought to positively regulate JA-mediated

responses in vivo (Devoto et al., 2002; Xu et al., 2002). The son1 mutant was

isolated in a screen for suppressors of the susceptible nim1-1 mutant (Kim

and Delaney, 2002). Mutant son1 plants exhibit constitutive resistance against

virulent fungal and bacterial pathogens without accompanying constitutive

expression of defence-related genes that are normally induced during

systemic acquired resistance. Thus, the F-box protein SON1 regulates a

novel induced defence response that is independent of both salicylic acid and

SAR (Kim and Delaney, 2002).

Supporting evidence for a role of the Ubiquitin/26S proteasome

pathway in plant immunity comes from the involvement of RAR1/SGT1 in a

broad range of race-specific resistance reactions to diverse pathogens (Austin

et al., 2002; Azevedo et al., 2002; Liu et al., 2002b; Peart et al., 2002; Tör et

al., 2002). In barley and N. benthamiana, SGT1 was shown to co-

immunoprecipitate with two SCF components, SKP1 and CULLIN1, (Azevedo

et al., 2002; Liu et al., 2002b). The same authors could also show that RAR1

physically associates with SGT1 in both barley and N. benthamiana plants

(Azevedo et al., 2002; Liu et al., 2002b). Further evidence supporting a link to

the Ubiquitin/26S proteasome pathway is based on the observation that both

RAR1 and SGT1 co-immunoprecipitate with the COP9 signalosome (CSN)

subunits in barley and tobacco (Azevedo et al., 2002; Liu et al., 2002b). It is

relevant that CSN has been shown to physically interact with the 26S

proteasome in vivo (Peng et al., 2003) and can interact with the SCF complex

General Discussion

100

through both the CULLIN and RBX1 subunits (Lyapina et al., 2001;

Schwechheimer et al., 2001; Cope et al., 2002). Therefore, one role of the

CSN is presumed to regulate SCF activity by removing ubiquitin-like

conjugates NEDD8 or RUB1 from the CULLIN subunit (Lyapina et al., 2001;

Schwechheimer et al., 2001). These recent findings strongly suggest

RAR1/SGT1 may connect the Ubiquitin/26S proteasome pathway to pathogen

responses in plants.

As discussed above, the data obtained from genetic and biochemical

analysis in barley collectively indicates that RAR1/SGT1 provides a link

between the Ubiquitin/26S proteasome pathway and MLA-mediated disease

resistance. However, questions that remain to be answered are: how does the

ubiquitynation pathway play a role in Mla-mediated resistance to the powdery

mildew pathogen? What are the targets for ubiquitynation and degradation in

MLA-mediated resistance responses?

SCF complexes may target suppressors in the Mla-triggered disease

resistance pathway for degradation via the 26S proteasome and thus

positively regulate defence responses. Potential suppressors can be host

factors that repress defence responses in the absence of pathogen. MLA-

mediated direct or indirect recognition of the cognate AVR effectors may

activate the ubiquitynation pathway to degrade the suppressors and hence

activate the down stream signalling cascade. In this context, the ubiquitin/26

proteasome pathway participates in MLA-mediated defence response

downstream of AVR recognition. As mentioned before, despite the unusually

high sequence similarity of known MLA R proteins, the resistance responses

can differ in their requirement for RAR1/SGT1. This might indicate that a

subset of Mla-mediated disease resistance responses may either occur

independently of ubiquitination or involves non-SCF-mediated ubiquitynation.

In this regard, it may be relevant that a second pool of HvSGT1 was detected

in barley leaf protein extracts, which associates with RAR1 and CSN subunits,

but not with SCF subunits (Azevedo, et al, 2002).

General Discussion

101

It is also conceivable that the MLA protein themselves are targets of

SCF complexes. Indirect evidence for this supposition comes from the rapid

disappearance of the CC-NB-LRR protein RPM1 coincident with the onset of

the HR (Boyes et al., 1998). This has been interpreted to restrict the extent of

cell death and overall resistance response at the site of infection. In yeast it

was shown that SGT1p physically interacts with another LRR-containing

adenylyl cyclase (CYR1; Dubacq et al., 2002; Schadick et al., 2002).

In the present study, yeast two-hybrid experiments using the fusion bait

containing the MLA1-LRR-CT identified HvSGT1 many times from the barley

prey library. Similarly, by targeted interaction analysis it was found that the

same bait interacts with both SGT1 isoforms present in Arabidopsis (AtSGT1a

and AtSGT1b; Shirasu K., JIC, personal communication). These findings are

consistent with the idea that MLA proteins are direct targets of SCF

complexes. The LexA and full-length MLA fusion proteins appeared to be

truncated at the C-terminal end in yeast (Fig. 10). For this reason it is at

present not possible to assess whether full-length MLA associates with

HvSGT1 in yeast.

7.3.2. RAR1/SGT1 may act as co-chaperones in MLA-mediated

resistance

New evidence revealed that the cytosolic heat shock protein 90

(HSP90) family play a crucial role in plant R proteins triggered immunity

(Hubert et al., 2003; Kanzaki et al., 2003; Liu et al., 2004; Lu et al., 2003;

Takahashi et al., 2003; reviewed in Schulze-Lefert, 2004). A mutant screen for

loss of AvrRpm1 recognition identified mutations of single amino acid

substitutions in the ATPase domain of HSP90.2, one of four HSP90 isoforms

in Arabidopsis plants expressing the RPM1 protein (Hubert, et al., 2003). By

assessing growth of various Avr containing P. parasitica strains on

Arabidopsis T-DNA insertion lines, it was demonstrated that a different

isoform, HSP90.1, is required for full RPS2 resistance function (Takahashi et

al., 2003). Enhanced susceptibility to the bacterial phytopathogen was also

General Discussion

102

seen upon injection of a known inhibitor of HSP90 activity, geldanamycin, into

leaves of RPS2 containing Arabidopsis. Virus-induce gene silencing (VIGS)

targeted at least four related NbHSP90 isoforms in Nicotiana benthamiana

and resulted in a loss of function of three tested NB-LRR proteins: RX, N and

PRF, mediating race-specific recognition of potato virus X (PVX), tobacco

mosaic virus (TMV) and P. syringae (expressing AvrPto effector), respectively

(Lu et al, 2003). Undoubtedly, the above findings demonstrate that the HSP90

family is essential for the function of multiple NB-LRR R proteins.

Heat-shock proteins are involved in folding and degradation of

damaged or misfolded peptides and assist in the correct folding of assembly

of protein complexes (Rutherford, 2003). Studies of HSP90 in animal and

yeast suggest that they work in association with many co-chaperones to

regulate activities of their substrates that are mainly involved in signalling

(Picard, 2002). The findings described above suggest that NB-LRR R proteins

might be HSP90 client proteins (Hubert et al., 2003; Takahashi et al., 2003;

Lu et al., 2003; Schulze-Lefert, 2003). Direct evidence for this claim comes

from the observation that HSP90 interacts with RPM1 in vivo and that steady-

state levels of RPM1 and RX are greatly reduced in HSP90 mutant or VIGS

induced HSP90 silenced plants, respectively (Hubert et al., 2003; Lu et al.,

2003).

Since animal RAR1 and SGT1 share a structural motif, the CS domain,

with a known co-chaperone, the p23 and HSP20/a-crystallin family, it has

been proposed that either protein might serve a co-chaperone-like role

together with HSP90 to regulate R protein activity and/or stability (Shirasu and

Schulze-Lefert, 2003; Schulze-Lefert, 2004; Garcia-Ranea et al., 2002).

Experimental evidence in support of this hypothesis is still limited:

RAR1/SGT1 co-immunoprecipitate with HSP90 in planta or can bind to

HSP90 in vitro and in vivo (Hubert et al., 2003; Takahashi et al., 2003; Liu et

al., 2004). The proposed co-chaperone role of RAR1/SGT1 may explain the

finding that RAR1 has a role in determining the steady-state levels of MLA

General Discussion

103

and RPM1 R proteins in planta. It may also explain that SGT1 physically

associates with several regulatory protein complexes in yeast and in plants

(Shirasu and Schulze-Lefert, 2003).

How can one explain the known differential engagement of

RAR1/SGT1 in MLA-mediated resistance responses to Bgh if RAR1/SGT1

exert a co-chaperone-like activity in race-specific immunity? Recent

biochemical evidence suggests that RAR1 increases protein abundance of

both MLA1 and MLA6 in barley (Bieri et al., unpublished data). MLA proteins

belong to the CC-NB-LRR type of intracellular R proteins and are highly

sequence related, sharing >90% sequence identity. Bearing this in mind, it

seems possible that RAR1 increases the abundance of all MLA proteins. In

one scenario, RAR1 might cooperate with HSP90 in assisting MLA to achieve

a proper conformation and to stabilize MLA protein by assembling it into a

complex. The different abundance of MLA1 and MLA6 detected in rar1 mutant

plants may reflect a quantitative difference in individual MLA protein

stability/or activity. By extrapolation from its role predicted in RPM1 and RX

protein function, SGT1 may also play a co-chaperone role in MLA protein

function although direct biochemical evidence is still lacking. However, the

role of SGT1 might be different from RAR1 in MLA mediated resistance. An

indication of this is the existence of a second pool of SGT1 containing SCF

subunits but not RAR1 in barley leaf extract (Azevedo et al., 2002). Likewise,

silencing of NbSGT1 does not reduce RX protein abundance (Lu et al., 2003).

7.4. Direct versus indirect AVRMLA recognition

The ‘gene-for-gene’ model provides a genetic framework for the phenomenon

of race-specific resistance in plants (Flor, 1971). The implication of gene-for-

gene relationships in plant and pathogen is the capacity of plants to recognize

pathogen-derived AVR effectors by R genes. However, several biochemical

interpretations could explain the genetic observations. The ‘receptor-ligand

model’ predicts that an R protein directly interacts with its cognate AVR

effector (Hammond-Kosack and Jones, 1997). The ‘guard hypothesis’

General Discussion

104

postulates that R proteins function in the surveillance of other host factors,

which in turn are targeted by AVR effectors (Dangl and Jones, 2001). Either

direct or indirect recognition by R proteins will trigger disease resistance

responses.

The Mla locus is unusually polymorphic, encoding about 30 resistance

specificities against Bgh isolates containing cognate AvrMla genes. The

experimentally validated intracellular localization of MLA proteins (Bieri S. and

S. Mauch, unpublished data) suggests that AVRMLA recognition occurs in the

cytoplasm. However, how MLA mediates AVRMLA recognition remains still

unclear. Transgenic barley lines expressing fully functional MLA1- or MLA6-

HA epitope tagged variants provide a powerful future tool to approach the

problem using biochemical methods. Preliminary evidence obtained by size

exclusion chromatography suggests that MLA assembles in a complex of

about 600-800 KDa in non-challenged plants (Mauch S., unpublished). One

possibility is that this complex contains other yet unknown host proteins.

Yeast two-hybrid screens using multiple LexA MLA fusion baits have identified

several barley proteins that are capable to physically associate with MLA

domains (see Chapter 6). One of these was shown to be HvSGT1.

Preliminary biochemical evidence suggest that this interaction occurs also in

planta because full-length MLA1-HA or MLA6-HA co-immunoprecipitate with

SGT1 (Mauch S., et al., MPIZ, unpublished). However, it remains to be shown

whether HSP90 and/or RAR1 are in the same complex. The characterization

of other potential components in presumed MLA complex(es) may provide

valuable hints for potential AVRMLA targets in planta.

Genetic mapping of Bgh AvrMla genes in the powdery mildew genome

revealed mainly dispersed and a few linked positions on multiple Bgh

chromosomes (Brown and Jessop, 1995; Caffier et al., 1996; Pedersen et al.,

2002). Recently, AvrMla10 and AvrMlk (the cognate Avr gene of the barley

Mlk R gene to Bgh) candidate genes were isolated from Bgh by map-based

cloning (Ridout C., et al., JIC, unpublished data). Sequence analysis of the

deduced AVRMLA10 and AVRMLK proteins reveals a conserved core region

General Discussion

105

and variable N- and C-termini. The availability of these Avr genes provides

future opportunities to study the AVRMLA-MLA interaction in the Bgh-barley

pathosystem in greater detail. Multiple AVR fusion baits are currently under

construction and will be used to screen the barley cDNA prey library. Any

interesting host factors interacting with the Bgh AVR proteins might contribute

to reveal the exact molecular mechanism of MLA-mediated AVRMLA

recognition.

A possible direct AVRMLA-MLA interaction can not be ruled out at this

stage. It was postulated that a direct AVR-R interaction may occur after

multiple folding switches of complexes containing R proteins (Schulze-Lefert,

2004). However, such folding switches might be transient and might not be

detectable by current biochemical techniques. A reconstitution of AVR-R

complexes in yeast and or Arabidopsis may provide a novel future tool to

unravel the mechanics of AVRMLA recognition. Known components for R

protein complex stability and/or function, e.g. HSP90, RAR1 and SGT1, might

be essential for such reconstitution experiments.

106

8. Summary

More than 30 race-specific resistance specificities to the powdery

mildew fungus Blumeria graminis f. sp. hordei (Bgh) map to the barley Mla

locus. This exceptionally polymorphic locus harbors multiple members of

three distantly related gene families, each encoding homologs of intracellular

disease resistance (R) proteins. Previously isolated Mla1 and Mla6 R genes

to Bgh encode highly sequence-related proteins (>90% identity) of

approximately 105 kD that contain an N-terminal coiled-coil (CC) structure, a

central nucleotide binding (NB) site, a Leucine rich repeat (LRR) region, and a

C-terminal non-LRR (CT) region. A subset of Mla R genes are known to

require for their function Rar1 and Sgt1. Intracellular RAR1 and SGT1 are

known to physically associate and are thought to have co-chaperone-like

activity.

It was hypothesized that other Mla R gene specificities might be closely

related to Mla1 and Mla6. A cosmid library was constructed from an Mla12-

containing barley line and cosmids containing candidate Mla12 genes were

isolated using molecular probes derived from Mla1. Race-specific resistance

activity to an AvrMla12 containing Bgh isolate was detected following transient

single cell expression of the Mla12 candidate gene in detached barley leaves.

This and the identification of point mutations in susceptible mla12 mutants

demonstrated that the candidate gene is Mla12. Over-expression of Mla12 in

the single cell assay shifted the slow Mla12 resistance response in wild type

plants to a rapid Mla1/Mla6-like resistance, terminating fungal growth at the

cell wall penetration stage before haustorium development. This indicates that

the cognate AvrMla12 product must be secreted before the switch from

surface to invasive fungal growth. Resistance mediated by Mla12 over-

expression retained the requirement for Rar1. Single cell dsRNAi gene

silencing experiments revealed a requirement of Mla12 resistance for Sgt1. A

series of reciprocal domains swaps between MLA1 and MLA6 identified in

Summary

107

each protein regions required for the recognition of cognate Bgh effectors

(encoded by AvrMla1 and AvrMla6). These regions comprise different but

overlapping LRR regions and the CT part. This is consistent with the finding

that the LRR and CT encoding parts of Mla1 and Mla6 exhibit evidence for

diversifying selection. Unexpectedly, MLA chimeras that confer AvrMla6

recognition specificity exhibit markedly different dependence on Rar1.

Furthermore, uncoupling of MLA6-specific resistance from RAR1 also

uncoupled the resistance response from SGT1. These findings suggest that

differences in the degree of RAR1 dependence of different MLA immune

responses are determined by intrinsic properties of MLA variants and place

RAR1/SGT1 activity downstream of and/or coincident with the action of

presumptive resistance protein–containing recognition complexes.

Previous analysis showed that loss-of-function mutations in both barley

and Arabidopsis Rar1 severely impair accumulation of MLA and RPM1 NB-

LRR proteins in healthy plants, respectively. In this study it was shown that

co-expression of RAR1 and MLA1 or MLA6 in heterologous yeast is not

sufficient to elevate MLA steady state levels. This suggests that RAR1

stabilizes MLA proteins indirectly, e.g. through other host factors that might be

components of MLA containing recognition complex(es). Yeast two-hybrid

screenings were used to identify host proteins directly interacting with MLA R

proteins. Several MLA interactors were identified using distinct or overlapping

domains from MLA1 or MLA6 proteins. Amongst those is the SGT1 protein

that is genetically required for the function of many NB-LRR proteins in

diverse plant species. Other MLA interactors exhibit high sequence

relatedness to carbonic anhydrase (CA), formin homology (FH) proteins, and

a WRKY transcription factor. Interestingly, tobacco carbonic anhydrase was

previously shown to be required for the activation of an efficient cell death

response triggered upon race-specific recognition of the Pseudomonas

syringae effector AVRPTO by the R gene Pto. The functional significance of

the identified interactors for Mla-mediated race-specific resistance to Bgh

remains to be validated, e.g. by dsRNAi experiments in the single cell gene

Summary

108

expression assay.

ZUSAMMENFASSUNG

In Gerste existieren über 30 rassespezifische Resistenz-Spezifitäten

gegen den Gerstenmehltau Blumeria graminis f. sp. hordei, die dem Mla-

Locus zugeordnet werden. An diesem außergewöhnlich polymorphen Locus

befindet sich eine Vielzahl von Genen aus drei entfernt verwandten Familien,

die Sequenzen mit hoher Homolgie zu bekannten Resistenzgenen

(Resistance gene homologs RGH) beinhalten.. Bisher isolierte Mla-Gene

kodieren für Proteine mit einem ungefähren Gewicht von 105 kD, die eine

bemerkenswert hohe Ähnlichkeit in Struktur und Sequenz (über 90%

identische Aminosäuren) zeigen. Die Proteine besitzen eine N-terminale

coiled-coil-Struktur (CC), eine zentrale Nukleotidbindestelle (NB), eine

Leucine-rich repeat-Region (LRR) und eine C-terminale nicht-LRR Region

(CT). Ein Teil der Mla R-Gene benötigt Sgt1 und Rar1um zu funktionieren.

Intrazelluläres SGT1 und RAR1 assoziieren physisch und haben

möglicherweise eine co-chaperone-ähnliche Aktivität.

Es wurde vermutet, dass andere Mla-Resistenzspezifitäten, zu den

bisher bekannten, Mla1 und Mla6, eng verwandt sind. Eine Cosmidbibliothek,

die von einer Mla12 enthaltenden Gerstelinie erstellt worden war, wurde

verwendet, um mit Hilfe von molekularen Sonden von Mla1 Cosmide mit

Kandidaten für Mla12 zu isolieren. Rassenspezifische Resistenzaktivität der

Mla12-Kandidaten gegen AvrMla12-enthaltendes Bgh-Isolat wurde durch

Überexpression mittels transienter Einzelzellgenexpression in abgetrennten

Blättern festgestellt. So und durch die Identifikation von Punktmutationen in

anfälligen mla12-Mutanten, wurde demonstriert, dass der Kandidat tatsächlich

Mla12 ist. Überexpression von Mla12 mittels transienter

Einzelzellgenexpression zeigte, dass die langsame Mla12-Immunantwort zu

Summary

109

einer schnellen, Mla1/6-ähnlichen Resistenz umgewandelt werden kann und

die Entwicklung des Pilzes vor der Ausbildung des Haustoriums, im Stadium

der Zellwandpenetration gestoppt wird. Das deutet an, dass die Sekretion des

korrespondierenden AvrMla12-Produkts vor dem‚ Umschalten von

oberflächlichem zu invasivem Wachstum stattfinden muss. Resistenz, die

durch Überexpression von Mla12 vermittelt wird, bleibt Rar1 abhängig. Eine

Abhängigkeit der Mla12-Resistenz von Sgt1 wurde durch transiente dsRNAi-

silencing-Versuche aufgedeckt.

Eine Serie von wechselseitigen Domänenaustauschexperimenten

zwischen MLA1 und MLA6 identifizierte in jedem Protein die Domäne, die für

die Erkennung der zugehörigen Effektormoleküle aus Mehltau (kodiert von

AvrMla1 und AvrMla6) notwendig ist. Diese Domänen lagen in verschiedenen,

aber überlappenden Teilen der LRR- und der CT-Region. Das stimmt mit

Hinweisen überein, dass diversifizierende Selektion in der LRR- und CT-

Region angreift. MLA-Chimären, die die spezifische Erkennung von

AVRMLA6 vermitteln, zeigten unerwarteterweise eine ausgesprochen

unterschiedliche Abhängigkeit von Rar1. Darüber hinaus wurde durch

Entkopplung der MLA6-spezifische Erkennung von RAR1 auch die

Abhängigkeit von SGT1 entkoppelt. Diese Ergebnisse legen nahe, dass der

unterschiedliche Grad der RAR1 Abhängigkeit verschiedener MLA-

Resistenzen, eine intrinsische Eigenschaft der verschiedenen MLA-Varianten

ist und platziert die Aktivität von RAR1/SGT1 hinter und/oder zusammen mit

mutmaßlichen R-Protein enthaltenden Komplexen.

Bisherige Studien haben gezeigt, dass Rar1 loss-of-function Mutanten

von Gerste und Arabidopsis in der Akkumulierung von MLA1

beziehungsweise RPM1 NB-LRR Proteinen in gesunden Pflanzen in stark

beeinträchtigt sind. In dieser Studie wurde gezeigt, dass die Co-Exprimierung

von RAR1 und MLA1 oder MLA6 in Hefe nicht ausreicht, um stationäre

Proteinmenge an MLA zu erhöhen. Dies lässt vermuten, dass RAR1 MLA

Proteine indirekt stabilisiert, z.B. durch andere Wirtsfaktoren, die Teil

eines/von MLA enthaltenden Erkennungskomplexes/n sein könnten. Yeast 2-

Summary

110

Hybrid Screening wurde für die Identifizierung von potentiellen Wirtsfaktoren

eingesetzt, die an der MLA-vermittelten Resistenz beteiligt sind. Mehrere

potentielle MLA-Interaktoren wurden für einzelne oder überlappende

Domänen von MLA1 oder MLA6 Proteinen identifiziert. Unter diesen

Interaktoren ist das SGT1-Protein, das für die Funktion von vielen NB-LRR-

Proteinen in verschiedenen Pflanzenarten notwendig ist, gefunden worden.

Andere MLA-Interaktoren zeigen hohe Sequenzverwandschaft zu

Carboanhydrase (CA), zu Proteinen mit Homologie zu Formin (FH), HvSGT1

und zu einem WRKY Transkriptionsfaktor. Interessanterweise wurde zuvor in

Tabak gezeigt, dass Carboanhydrase für die Aktivierung einer effizienten

Zelltodantwort nach rassespezifischer Erkennung des Pseudomonas syringae

Effektors AVRPTO durch das R Gene Pto notwendig ist. Die funktionelle

Signifikanz der identifizierten Interaktoren für die Mla-vermittelte

rassenspezifische Resistenz gegen Bgh muss noch bestätigt werden, z.B.

durch dsRNAi-Experimente mittels transienter Einzelzellgenexpression.

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10. Appendix: Publications

The Plant Cell, Vol. 15, 732–744, March 2003, www.plantcell.org © 2003 American Society of Plant Biologists

Recognition Specificity and RAR1/SGT1 Dependence in Barley

Mla

Disease Resistance Genes to the Powdery Mildew Fungus

Qian-Hua Shen,

a,b,1

Fasong Zhou,

b,1,2

Stephane Bieri,

a,1

Thomas Haizel,

b

Ken Shirasu,

b

and Paul Schulze-Lefert

a,b,3

a

Max-Planck-Institut für Züchtungsforschung, Department of Plant–Microbe Interactions, Carl-von-Linné-Weg 10, D-50829 Köln, Germany

b

The Sainsbury Laboratory, John Innes Centre, Colney Lane, NR4 7UH Norwich, United Kingdom

A large number of resistance specificities to the powdery mildew fungus

Blumeria graminis

f. sp.

hordei

map to the barley

Mla

locus. This complex locus harbors multiple members of three distantly related gene families that encode proteins thatcontain an N-terminal coiled-coil (CC) structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and aC-terminal non-LRR (CT) region. We identified

Mla12

, which encodes a CC-NB-LRR-CT protein that shares 89 and 92%identical residues with the known proteins MLA1 and MLA6. Slow

Mla12

-triggered resistance was altered dramatically to arapid response by overexpression of

Mla12

. A series of reciprocal domains swaps between MLA1 and MLA6 identified ineach protein recognition domain for cognate powdery mildew fungus avirulence genes (

AvrMla1

and

AvrMla6

). These do-

mains were within different but overlapping LRR regions and the CT part. Unexpectedly, MLA chimeras that confer

AvrMla6

recognition exhibited markedly different dependence on

Rar1

, a gene required for the function of some but not all

Mla

resistance specificities. Furthermore, uncoupling of MLA6-specific function from RAR1 also uncoupled the responsefrom SGT1, a protein known to associate physically with RAR1. Our findings suggest that differences in the degree of RAR1dependence of different MLA immunity responses are determined by intrinsic properties of MLA variants and place RAR1/SGT1 activity downstream of and/or coincident with the action of resistance protein–containing recognition complexes.

INTRODUCTION

Intraspecific genetic variation in the capacity of plants to com-bat microbial attack is confined mainly to disease resistance (

R

)loci. These can encode a single gene but frequently they arecomplex, harboring multiple similar and/or dissimilar

R

genes(reviewed by Ellis et al., 2000). A single

R

gene has the capacityto recognize one or very few normally unrelated strain-specificpathogen effector molecules (encoded by avirulence [

Avr

]genes) that are released during pathogenesis. Most

R

genesencode one of two groups of Leu-rich repeat (LRR)–containingproteins. An intracellular class shares a central nucleotide bind-ing (NB) site and C-terminal LRRs with variable repeat num-bers. This is the largest group of known R proteins and can bedivided further into subfamilies containing either N-terminalsequences predicted to form a coiled-coil (CC) structure (CC-NB-LRR subfamily) or sequences that are related to the cyto-plasmic domain of the

Drosophila

Toll and human Interleukin1receptor (TIR-NB-LRR). A second R protein class is membrane-anchored by a single transmembrane helix, consists of variablerepeat numbers of extracellular LRRs, and contains at least in

one case an intracellular Ser/Thr kinase module (reviewed byEllis et al., 2000).

Little is known about the molecular mechanics of the R-AVRrecognition process. Recent studies suggest that members ofthe intracellular and membrane-anchored classes assemble inpreformed heteromultimeric recognition complexes in the ab-sence of pathogens (Leister and Katagiri, 2000; Holt et al.,2002; Mackey et al., 2002; Rivas et al., 2002a, 2002b). With oneexception, there are no documented examples of direct inter-actions between LRR-containing R and AVR proteins (rice Pi-taand AVR-Pita from

Magnaporthe grisea

; Jia et al., 2000). Thus,it seems possible that R proteins function indirectly in the rec-ognition process, which involves the surveillance of a host pro-tein or a complex that is targeted by

AVR

products (Dangl andJones, 2001; Mackey et al., 2002).

Approximately 30 genetically characterized barley

Mla

vari-ants have been described, each triggering immunity responsesupon recognition of cognate isolate-specific powdery mildewfungus (

Blumeria graminis

f. sp.

hordei

[

Bgh

]) effector mole-cules (encoded by

AvrMla

genes) (Jørgensen, 1994). Some ofthese variants confer a rapid resistance response resulting in

Bgh

growth termination at an early stage during pathogenesis,whereas others trigger a delayed response that permits sub-stantial growth of fungal hyphae on the leaf surface (Wise andEllingboe, 1983; Boyd et al., 1995). Although none of the

BghAvrMla

genes has been isolated to date, their genetic mappingin the powdery mildew genome revealed mainly dispersed anda few linked positions on multiple

Bgh

chromosomes (Brownand Jessop, 1995; Caffier et al., 1996; Pedersen et al., 2002).

1

These authors contributed equally to this work.

2

Current address: Boyce Thompson Institute for Plant Research, CornellUniversity, Tower Road, Ithaca, NY 14853.

3

To whom correspondence should be addressed. E-mail [email protected]; fax 49-221-5062313.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.009258.

Functional Dissection of

Mla

Resistance 733

The complex

Mla

locus was located genetically and physicallywithin an interval of

�

250 kb (Wei et al., 1999). A contiguousDNA sequence of the interval in barley cv Morex revealed 32predicted genes, of which 8 encode CC-NB-LRR resistancegene homologs (

RGHs

) (Wei et al., 2002). The

RGHs

belong tothree dissimilar families sharing

�

43% amino acid sequencesimilarity between families (Wei et al., 1999, 2002). BecauseMorex lacks a known

Mla

resistance specificity, the first twoidentified

Mla

powdery mildew

R

genes,

Mla1

and

Mla6

, wereisolated from other barley accessions (Halterman et al., 2001;Zhou et al., 2001). The deduced proteins share 91% identicalresidues and show highest overall similarity to the deducedMorex

RGH1bcd

family member (83 and 79% identity to MLA1and MLA6, respectively) (Halterman et al., 2001; Wei et al.,2002).

Mutants of barley

Rar1

were isolated originally as suppres-sors of

Mla12

function, and wild-type

Rar1

was shown subse-quently to be required for the function of a subset of

Mla

pow-dery mildew resistance specificities (e.g.,

Mla6

and

Mla12

butnot

Mla1

) (Torp and Jørgensen, 1986; Jørgensen, 1996). Ho-mologs of

Rar1

in Arabidopsis and

Nicotiana benthamiana

playa conserved role in the function of a subset of NB-LRR R pro-teins that confer resistance to different pathogens (Liu et al.,2002a; Muskett et al., 2002; Tornero et al., 2002). The highlyconserved zinc binding RAR1 proteins interact physically withanother conserved protein, SGT1, which was demonstratedoriginally to function in ubiquitin-dependent cell cycle control inyeast (Kitagawa et al., 1999; Shirasu et al., 1999a; Azevedo etal., 2002; Liu et al., 2002b). Genetic evidence for a role of plant

SGT1

in

R

gene–triggered resistance was obtained from Arabi-dopsis

sgt1b

mutants and

SGT1

gene-silencing experiments inbarley and

N. benthamiana

(Austin et al., 2002; Azevedo et al.,2002; Liu et al., 2002b; Peart et al., 2002; Tör et al., 2002). Bar-ley and

N. benthamiana SGT1

associate physically with one orseveral SCF ubiquitin E3 ligase complexes and the COP9 sig-nalosome (Azevedo et al., 2002; Liu et al., 2002b). Becausegene silencing of the core SCF component, SKP1, or the COP9signalosome compromised

R

gene–triggered resistance in

N.benthamiana

, it seems likely that ubiquitin-protein conjugation path-ways play an important role in plant innate immunity responses(Liu et al., 2002b). However, it remains unclear whether ubiq-uitin-dependent processes occur upstream of, coincident with,or downstream of R protein–containing recognition complexes.

Here, we exploited a high sequence relatedness betweenidentified (

Mla1

and

Mla6

) and other genetically characterized

Mla

specificities to clone

Mla12

. Although

Mla12

might be anallele of

Mla1

and

Mla6

, it differs from them by belonging to asubgroup of

Mla

variants that trigger delayed resistance re-sponses (Freialdenhoven et al., 1994; Boyd et al., 1995). Usinga single-cell transient gene expression assay (Shirasu et al.,1999b; Zhou et al., 2001), we demonstrate that

Mla12

overex-pression shifts the slow

Mla12

-triggered response to a rapid

Mla1

/

Mla6

-like resistance. We gained insights into structure-function relationships of MLA proteins by analyzing a series ofreciprocal domain swaps between MLA1 and MLA6. This anal-ysis revealed a function for the MLA LRR-CT unit in specificitydetermination, whereas CC-NB and LRR sequences modulatedRAR1 dependence. Moreover, we show that recognition speci-

ficity can be uncoupled from both RAR1 and SGT1 dependence.We discuss possible roles of RAR1/SGT1 in folding presumedMLA recognition complexes and in signaling downstream ofactivated recognition complexes.

RESULTS

Isolation of

Mla12

and Characterization of Susceptible Mutant Alleles

To isolate

Mla12

, we constructed a genomic cosmid librarycomprising five barley genome equivalents using DNA from cvSultan 5 containing

Mla12

(see Methods). Sixteen cosmidclones were isolated from this library with a DNA probe corre-sponding to the LRR region of MLA1. Low-pass DNA sequenc-ing of the cosmid clones revealed that all of them contain NB-LRR–type

RGHs

. Two clones, designated Sp14-1 and Sp14-4,contain identical

RGHs

showing

�

90% sequence identity to

Mla1

and

Mla6

in deduced exon and intron sequences. A closercomparison of the

NB-LRR

gene in Sp14-4 with

Mla1

and

Mla6

revealed an identical 5

�

untranslated small open reading frame ofnine amino acids and the same intron-exon structure (Haltermanet al., 2001; Zhou et al., 2001). These genes share a simple se-quence repeat (AT)

n

in intron 3, although the exact numbers ofthe repeats differ (see Figure 6 below). Therefore, we consid-ered the

RGH

in Sp14-4 a candidate

Mla12

gene that encodesa predicted CC-NB-LRR-CT protein of 108 kD sharing 89%identical residues with MLA1 and 92% identical residues withMLA6 (Figure 1).

To obtain evidence for the function of the candidate

R

gene,we took advantage of chemically induced susceptible mutantsthat were isolated previously from a mutagenized barley M

2

population of Sultan 5 harboring

Mla12

(Torp and Jørgensen,1986; Jørgensen, 1988). Genetic analysis indicated that sus-ceptibility in some of the mutants (e.g., mutants M66 and M86)is likely attributable to mutations in

Mla12

, whereas susceptibil-ity in three other lines (M22, M82, and M100) resulted from ex-tragenic suppressor mutations of

Mla12

function. Mutant linesM82 and M100 were demonstrated to contain recessive muta-tions in

Rar1

(

rar1-1

and

rar1-2

, respectively) (Shirasu et al.,1999a), and genetic analysis of mutant M22 suggested anothergene required for

Mla12

function, designated

Rar2

(Jørgensen,1988, 1996; Freialdenhoven et al., 1994). DNA sequence analy-sis of the candidate

Mla12

in the susceptible mutants M66 andM86 revealed in each a single nucleotide substitution com-pared with the wild-type gene. The substitutions replace aminoacid Leu-631 with Arg in the second LRR of the deduced can-didate MLA12 protein in M66 and amino acid Glu-866 with Lysin the CT region in M86, respectively (Figure 1). Thus, we con-cluded that the Sp14-4–derived candidate gene probably is

Mla12

.DNA marker–based mapping of susceptibility conferred by

the M22 mutant revealed its location on chromosome 1H at the

Mla

locus between restriction fragment length polymorphismmarkers MWG036 and MWG068 (Schüller et al., 1992; our un-published data). This finding suggested that susceptibility mightbe caused by a mutation in

Mla12

or in a tightly linked gene.DNA sequence analysis of the candidate

Mla12

in M22 plants

734 The Plant Cell

revealed a single nucleotide substitution that replaces aminoacid Lys-916 with Met in the CT region (Figure 1). This findingsuggests that M22, like M66 and M86, likely is a mutant alleleof

Mla12

(see below).

Overexpression of

Mla12

Alters the Resistance Kinetics but Retains

Rar1

Dependence

To test directly the function of the candidate

Mla12

gene,Sp14-4 DNA was delivered into epidermal cells of detachedbarley leaves by particle bombardment (Shirasu et al., 1999b).Transformed cells were tested for their ability to activate race-specific powdery mildew resistance upon inoculation with

Bgh

conidiospores of isolates expressing or lacking

AvrMla12 (iso-late A6 harboring AvrMla6 and AvrMla12 and isolate K1 har-boring AvrMla1) (Zhou et al., 2001). Infection phenotypes oftransgene-expressing epidermal cells were microscopically in-spected at 48 h after inoculation by scoring the presence or ab-

sence of intracellular Bgh haustoria at single interaction sites.Unlike control bombardments with cosmid DNA harboring Mla1or Mla6, which are known to mediate race-specific resistancein the transient gene expression assay (Halterman et al., 2001;Zhou et al., 2001), delivery of Sp14-4 DNA failed to trigger de-tectable resistance upon inoculation with Bgh strains A6 andK1 (data not shown). This effect may be caused by insufficient5� flanking regulatory sequences (�400 bp upstream of thetranscription start) in cosmid Sp14-4, driving expression of thecandidate Mla12, or delayed activation of Mla12 compared withMla1 and Mla6 resistance (see Discussion) (Freialdenhoven etal., 1994; Boyd et al., 1995).

To examine this possibility further, we subcloned the codingregion of the Mla12 candidate under the control of the strongmaize ubiquitin promoter and the nopaline synthase (Nos) ter-minator. DNA of this overexpression construct and two similarcontrol overexpression plasmids harboring Mla1 or Mla6 weredelivered into leaf epidermal cells of barley cv Ingrid lacking

Figure 1. Amino Acid Sequence Alignment of Deduced Products of the Mla1, Mla6, and Mla12 Genes.

Residues identical to those in MLA1 are shown as dots, and deletions are shown as hyphens. A predicted CC structure is underlined. The starts of theNB, LRR, and CT regions are indicated with arrows and are operational according to Zhou et al. (2001). Boldface letters in the NB domain indicateamino acid motifs conserved among known NB-LRR proteins. Boxes indicate amino acid exchanges identified in three susceptible Mla12 mutants,and affected residues are shaded in black.

Functional Dissection of Mla Resistance 735

Mla12 and Mlo (Figure 2A). Delivery of each plasmid DNA to-gether with an Mlo-expressing construct resulted in a hausto-rium index of 2 to 5% upon challenge with the Bgh isolate con-taining the cognate Avr genes, whereas the control compatibleinteractions showed an index of �80%. Note that the very highlevel of haustorium incidence found in the compatible interac-tions likely is the result of cobombardment of the race-nonspe-cific defense modulator Mlo, which renders transformed epi-dermal cells supersusceptible to the fungus (Kim et al., 2002).These data provided evidence that the candidate Mla12 genesubcloned from cosmid Sp14-4 triggered AvrMla12-dependentBgh growth termination. Interestingly, bombardments with empty

vector DNA into epidermal cells of Sultan 5, which containsMla12, resulted in a high haustorium index of 45% when inocu-lated with the incompatible isolate Bgh A6 (Figure 2B). Thisfinding suggests that Mla12 resistance is not effective beforehaustorium development, consistent with a previous quantita-tive inspection of single interaction sites in resistant Mla12wild-type and susceptible mutant leaves (Freialdenhoven et al.,1994). However, when the putative Mla12 was overexpressed inSultan 5 using the single cell expression assay, the haustoriumindex was reduced to �2%, similar to the level conferred byMla6 (Figure 2B). Apparently, overexpression of the candidateMla12 shifted the resistance response from posthaustoriumgrowth arrest to an abortion of fungal development beforehaustorium formation.

To corroborate the function of Mla12, we bombarded theoverexpression construct in epidermal cells of mutant linesM66, M22, and M100 (the latter contains the severely defectiverar1-2 allele) (Shirasu et al., 1999a) (Figure 2C). In these experi-ments, full AvrMla12-dependent resistance was restored inboth M66 and M22 plants, demonstrating that the mutant phe-notypes were complemented by the candidate Mla12. By con-trast, neither overexpression of Mla6 nor overexpression of thecandidate Mla12 restored full resistance in the rar1-2 mutantline M100. The Mla12 overexpression phenotype was affectedmore strongly than the Mla6 response in the rar1 mutant back-ground. Together, these data strongly support our claim thatthe RGH in cosmid Sp14-4 is Mla12.

Context-Dependent Function of Conserved MLA Residues Leu-631 and Lys-916

We noted that amino acid substitutions in the susceptibleMla12 mutants M66 (L631R) and M22 (K916M) affect residuesthat are conserved in MLA1 and MLA6, whereas the substitu-tion in mutant M86 (E866K) changes a nonconserved residue(Figure 1). To investigate the importance of Leu-631 and Lys-916 in Mla1- and Mla6-triggered resistance, the same aminoacid substitutions were introduced into Mla1 and Mla6 underthe control of the ubiquitin promoter and were reintroducedinto Mla12 for comparison and confirmation. Wild-type andmutant variant plants were tested in the transient gene expres-sion system. This analysis showed that Mla12 mutant variantL631R impaired AvrMla12-dependent resistance fully (84%)and K916M impaired it partially (31%), indicating that theMLA12 (K916M) variant protein retains residual activity (Figure3). This observation is consistent with the fully compromisedand partially impaired Mla12 resistance reported for M66 andM22 mutant plants (infection types 4 and 2/3, respectively)(Torp and Jørgensen, 1986) and validates the usefulness of thesingle-cell assay to evaluate Mla12 activity using the strongubiquitin promoter. The weakly susceptible infection phenotypeof M22 mutant plants likely complicated the scoring of infectionphenotypes in progeny derived from M22 test crosses and mayexplain the apparent misinterpretation of the mutant line as anextragenic suppressor of Mla12 resistance (Jørgensen, 1988,1996). Surprisingly, despite an overall sequence relatedness of90% between the tested MLA proteins, none of the amino acidreplacements in MLA6 or MLA1 resulted in a detectable change

Figure 2. Complementation of Susceptible Mla12 Mutants by Overex-pression of Mla12 Resistance.

Relative single cell resistance/susceptibility upon delivery of various Mlatransgenes at 48 h after spore inoculation is indicated by haustorium in-dices of attacked �-glucuronidase (GUS)–expressing cells (%). Datashown were obtained by bombardment of plasmid DNAs into epidermalcells of detached barley leaves (described by Shirasu et al., 1999b;Zhou et al., 2001). A �-glucuronidase reporter gene was used to identifytransformed cells.(A) The indicated transgenes were tested in detached leaves of barleycv Ingrid harboring mlo-3 Rar1. In this line, broad-spectrum mlo-3 resis-tance was complemented by cobombardment with a plasmid express-ing wild-type Mlo; this renders cells supersusceptible to all tested Bghisolates (Zhou et al., 2001; Kim et al., 2002). Results obtained with theBgh isolate K1 (AvrMla1) are shown by closed columns, and results ob-tained with isolate A6 (AvrMla6 and AvrMla12) are shown by open col-umns in downward orientation. The data shown are means of at leastthree independent experiments (SD indicated). Each experiment in-volved light microscopic examination of at least 100 interaction sitesbetween a single Bgh sporeling and an attacked epidermal cell.(B) The indicated transgenes and an empty vector control were deliv-ered into epidermal cells of Sultan 5 containing Mla12 Mlo Rar1. Experi-mental conditions and symbols are identical to those in (A).(C) Transgene Mla12 or an empty vector control was delivered into epi-dermal cells of two susceptible Mla12 mutant lines (M66 and M22).Transgene Mla6 or Mla12 or an empty vector control also was deliveredinto the rar1-2 mutant line M100. Experimental conditions and symbolsare identical to those in (A).

736 The Plant Cell

of resistance activity compared with that in the respective wild-type genes (Figure 3). Thus, it is possible that other regions arecritical for R protein function in MLA1 and MLA6 (see below).Alternatively, other residues that are absent or polymorphic inMLA12 might compensate for the functional contributions ofLeu-631 and Lys-916 in the MLA1/MLA6 substitution mutants.

Recognition Specificity Is Determined by the LRR-CT Unit

For further analysis of regions that are critical for MLA function,we constructed a series of reciprocal domain swaps betweenMla1 and Mla6 (Figure 4A). These two R genes recognize differ-ent AvrMla genes and have different requirements for Rar1 andSgt1 (Halterman et al., 2001; Zhou et al., 2001; Azevedo et al.,2002). The maize ubiquitin promoter drove the expression ofeach chimeric gene, and their function was tested after bom-bardment into leaf epidermal cells by spore inoculation withBgh isolates K1 (AvrMla1) and A6 (AvrMla6) at 15 h after deliv-ery. Recognition specificity and activity of the chimeras werecompared with those of the respective Mla1 and Mla6 wild-typegenes whose expression was driven by either native regulatory5� sequences or the strong ubiquitin promoter (Figure 4B). Nosignificantly different activity was seen using constructs drivenby the native or the strong ubiquitin promoter. Full AvrMla6-dependent recognition specificity was retained in chimerascontaining the complete MLA1-derived CC-NB domains and inchimeras containing progressively more MLA1-derived N-ter-

minal LRR repeats (constructs 16666, 11666, and 11166; Figure4B). Activities mediated by chimeras containing only MLA6-derived LRRs 3 to 11 (11661) or only the MLA6-derived C ter-minus (11116) were inactive or severely impaired, respectively.These data suggest that MLA6 LRRs 9 to 11 act together withthe cognate C-terminal domain to confer AvrMla6 recognitionspecificity.

Reciprocal domain swaps showed that AvrMla1-dependentactivity was retained upon replacement of the entire MLA1 CC-NB domain only and upon additional replacement of LRRs 1and 2 (constructs 61111 and 66111). Interestingly, longer sub-stitutions up to LRR 8 rendered the 66611 chimera fully inac-tive, although the reciprocal construct 11166 fully retainedAvrMla6-dependent activity. Substitutions containing LRRs 3 to11 (construct 11661) also compromised AvrMla1 recognitionspecificity. Because chimeras containing only MLA1-derivedLRRs 3 to 11 (66116) or only the MLA1-derived C terminus(66661) were inactive, we conclude that MLA1-derived LRRs 3to 11 together with the cognate C-terminal domain are requiredfor MLA1 recognition specificity.

Uncoupling MLA6 Recognition Specificity fromRAR1 Dependence

Barley Rar1 is required for the function of Mla6 but not Mla1(Jørgensen, 1996; Halterman et al., 2001; Zhou et al., 2001).This fact prompted us to examine the activities of wild-typeMLA1 and MLA6 and the MLA chimeras in the rar1-2 geneticbackground (Figure 4C). The rar1-2 mutation leads to a tran-script-splicing defect, and a RAR1 antiserum fails to detectRAR1 signals on protein gel blots (Azevedo et al., 2002). Deliv-ery of wild-type MLA1 or MLA6 plasmid DNA in rar1-2 leaf epi-dermal cells led to fully retained or partially compromised resis-tance (4 and 39% haustorium index, respectively) (Figure 4C).No significant differences were found between wild-type con-structs driven by the native and strong ubiquitin promoters.Thus, Mla6 function is compromised partially by the rar1-2 mu-tation compared with bombardments of the same constructs inthe Rar1 background (Figure 4B). Remarkably, delivery of thethree chimeras conferring AvrMla6-dependent resistance inRar1 plants (16666, 11666, and 11166) displayed either fullRAR1 dependence (constructs 16666 and 11666, each show-ing 80% haustorium index) or uncoupled RAR1 dependencefrom recognition specificity (construct 11166, showing 10%haustorium index) in the rar1-2 background. Neither of the twochimeras that retained AvrMla1-dependent resistance activity(61111 and 66111) was impaired functionally upon delivery inrar1-2 mutant plants. Unless MLA6 accumulation is self-limited,we conclude that RAR1 dependence cannot be overcome byMla6 overexpression and appears to be modulated by both theCC-NB and LRR regions. Because it was reported that an Ara-bidopsis rar1 mutant line failed to accumulate a CC-NB-LRRprotein, RPM1 (Tornero et al. 2002), we also tested whetherMLA6 becomes unstable in the rar1-2 mutant background. At96 h after delivery, MLA6 remained as active as at 15 h afterdelivery (39% haustorium index), suggesting that the stability ofMLA6 remained unchanged in rar1-2 plants (see below for ex-amples of unstable MLA variants 16666 and 11666).

Figure 3. Context-Dependent Functions of Conserved MLA ResiduesLeu-631 and Lys-916.

Mean values of single cell resistance/susceptibility (%) are shown at leftafter delivery of Mla1, Mla6, or Mla12 into the genetic background of cvIngrid (mlo-3 Rar1). Results obtained with L631R variants of Mla1, Mla6,and Mla12 are shown in the middle. Results obtained with Mla1, Mla6,and Mla12 variants each containing a K to M substitution at the indi-cated positions are shown at right. Experimental conditions and desig-nations are identical to those in Figure 2. GUS, �-glucuronidase.


Requirement of Sgt1 for MLA-Mediated Resistance

Barley Sgt1 (HvSgt1) was shown to be required for Mla6- butnot Mla1-mediated resistance using double-stranded RNA in-terference (dsRNAi) gene silencing of HvSgt1 in a single-cellexpression system (Azevedo et al., 2002). This technique wasused to examine in the Rar1 wild-type background the SGT1requirement of MLA chimeras that retain MLA6 recognitionspecificity (constructs 16666, 11666, and 11166 in Figure 5). Inthese experiments, Bgh spore inoculations were performed at48 or 96 h after delivery, and the leaf tissue was fixed for micro-scopic analysis 48 h after spore inoculation. Cobombardmentof SGT1 dsRNAi DNA with a plasmid driving wild-type Mla6from the ubiquitin promoter resulted in a small but significantlyincreased haustorium index (19% at 96 h after delivery) com-pared with delivery of an empty dsRNAi vector control (2%).This finding is consistent with previous data (Azevedo et al.,2002). Unexpectedly, the functioning of chimeras 16666 and11666 was partially impaired at 48 h after delivery in cobom-bardment experiments with the empty vector dsRNAi control.This phenomenon was time dependent in that the chimeraswere almost completely inactive at 96 h after delivery. This find-ing may indicate that the two chimeric MLA proteins are lessstable or that fewer or less active recognition complexes areformed compared with complex formation in the MLA6 wild-

type protein. Nevertheless, at 48 h after delivery, cobombard-ment of plasmids 16666 and 11666 with SGT1 dsRNAi DNAsignificantly enhanced the haustorium index compared withthat in empty vector controls (P � 0.05), indicating at least apartial requirement of the chimeras for Sgt1. By contrast, the11166 chimeric protein retained full activity upon cobombard-ment with the empty dsRNAi plasmid control, and its functionremained unaffected by Sgt1 silencing even at 96 h after deliv-ery (Figure 5). Unlike wild-type Mla6, AvrMla6-dependent resis-tance conferred by the 11166 variant appears to be uncoupledfrom both Rar1 and Sgt1 dependence (Figures 4C and 5).

DISCUSSION

Allelic Variants Encode MLA Powdery Mildew R Proteins

Eight NB-LRR genes are present in a 260-kb interval compris-ing the Mla locus in barley cv Morex and were classified intothree dissimilar families (RGH1, RGH2, and RGH3) with �43%amino acid sequence similarity between families (Wei et al.,2002). Computational analysis of the Morex 260-kb sequencecontig suggested that a progenitor Mla locus harbored at �8million years before the present one member of each RGH fam-ily (RGH1bcd, RGH2a, and RGH3a) (Wei et al., 2002). Each of

Figure 4. Domain Swaps between MLA1 and MLA6 Reveal Determinants for Recognition Specificity and RAR1 Dependence.

(A) Schemes of MLA6 (yellow), MLA1 (green), and 10 chimeras are shown. The relative positions of the CC, NB, LRR, and CT parts are indicated attop, and acronyms for each chimera are shown at left. The stars indicate gene expression driven by native 5� flanking sequences; the strong ubiquitinpromoter drove the expression of all other genes.(B) All genes shown in (A) were expressed in the Rar1 wild-type background, and mean values of single cell resistance/susceptibility were scored mi-croscopically upon challenge inoculation with Bgh isolates A6 or K1. Experimental conditions and designations are identical to those in Figure 2.GUS, �-glucuronidase.(C) All genes shown in (A) were expressed in the rar1-2 mutant background, and mean values of single cell resistance/susceptibility were scored mi-croscopically upon challenge inoculation with Bgh isolates A6 or K1. Experimental conditions and designations are identical to those in Figure 2.

738 The Plant Cell

the Mla powdery mildew R genes identified to date shows high-est overall sequence similarity to Morex RGH1bcd in coding re-gions and shares the same exon/intron structure (Figure 6) (Weiet al., 2002). Unlike RGH1bcd, however, Mla1/6/12 each con-tains a 5� untranslated open reading frame and, within intron 3,an (AT)n simple sequence repeat consisting of different repeatnumbers (Figure 6). Also, Morex RGH1bcd contains a BARE1solo long terminal repeat in intron 3 that is absent in Mla1/6/12,and the presence of a 29-bp deletion in the LRR region, result-ing in a premature stop codon, suggests that it is nonfunctional(Figure 6). Because Morex lacks known Mla powdery mildewresistance specificity, it has been inferred that RGH1bcd is anaturally inactive allele of Mla1 and Mla6 that may have servedas a progenitor for the other Morex RGH1 family members(RGH1a, RGH1e, and RGH1f ) (Wei et al., 2002). Closer exami-nation of all possible pair-wise sequence comparisons of thefour Morex RGH1 variants and the identified Mla resistancespecificities revealed for exon 4 sequences a common clusterthat includes genes Mla1/6/12 and RGH1bcd. However, se-quences of RGH1bcd exon 3 and intron 3 cluster together withthe other RGH1 gene sequences, whereas the identified Mla re-sistance specificities form a second group (even after theexclusion of the BARE1 long terminal repeat in intron 3 ofRGH1bcd; data not shown). Therefore, it is possible thatRGH1bcd is the product of a recombination between an ances-tral Morex allele of Mla1/6/12 and another more divergent RGH.

DNA gel blot analysis and preliminary sequence informationobtained from nearly isogenic barley lines containing other Mlapowdery mildew resistance specificities indicate for each linethe presence of one candidate gene with high sequence relat-edness to MLA1/6/12 (data not shown). Thus, it is possible thatmany genetically characterized powdery mildew R genes at Mlaare variants of the same ancestral RGH1 family member. Thepresence of the (AT)n microsatellite in all Mla R genes in Bghidentified to date and its absence in currently available MlaRGHs are consistent with our hypothesis, because recent find-ings indicate that most microsatellites reside in regions predat-ing recent genome expansion in plants (Morgante et al., 2002).

The very high level of DNA sequence conservation in exonand intron sequences of identified Mla R genes (average overallidentity of 94 and 93%, respectively) may be indicative of se-lective constraints acting on both coding and noncoding regions.By contrast, inspection of flanking regions revealed evidencefor extensive intralocus recombination events that reshuffledboth genes and intergenic regions (Figure 6). For example, aHORPIA2 element was found in the same direction immediately3� of RGH1bcd and 3� of Mla1, whereas 3.7 kb of 3� flankingsequence of Mla6 showed no significant relatedness to anystretch in the 260-kb Mla Morex contig. Sequences located im-mediately 3� of Mla12 were found 5.5 kb downstream ofRGH1bcd, indicating an extensive intralocus insertion/deletionevent. Morex RGH1f/e exhibited highest sequence relatednessto the Mla1 paralog Mla1-2; their altered relative orientation toRGH1bcd and Mla1, respectively, suggests the occurrence ofan intralocus inversion event (Figure 6).

Altering Resistance Response Kinetics by Mla Dosage

Different Mla resistance genes to Bgh show characteristic in-fection phenotypes that are macroscopically visible by differentinfection types (Boyd et al., 1995). A quantitative analysis ofsingle interaction sites in nearly isogenic lines containing differ-ent Mla genes revealed for Mla1 and Mla6 early termination ofBgh growth coincident with haustorium differentiation (Boyd etal., 1995). By contrast, Mla3 and Mla7 mediated cessation offungal growth at a later stage of the infection process, permit-ting the growth of elongating secondary hyphae on the leaf sur-face in addition to haustorium differentiation. These Mla gene-specific differences correlated with the timing of a cell deathresponse that was either rapid, involving attacked epidermalcells, or slower, including epidermal and subtending mesophyllcells (Boyd et al., 1995). Similarly, delayed cell death–associ-ated resistance is characteristic for lines carrying Mla12, per-mitting indistinguishable fungal growth for up to 36 h after Bghspore inoculation and a high haustorium index of �60% onboth Mla12-resistant and Mla12-susceptible mutant plants(Freialdenhoven et al., 1994). It is possible that differences in thespeed of Mla resistance responses are the indirect consequenceof different infection stage–specific delivery systems for particu-lar Bgh AVRMLA effector proteins (e.g., delivery of AVRMLA12only after or coincident with haustorium differen-tiation).

Precedence for this idea is found in the expression of Clado-sporium fulvum AVR9, which is induced strongly upon a switchfrom surface to intercellular growth of the fungus in leaves,

Figure 5. Single Cell Silencing of Sgt1 by dsRNAi.

Wild-type Mla6 or chimeras retaining AvrMla6-dependent recognitionspecificity were coexpressed with a HvSgt1 dsRNAi-silencing plasmid(Azevedo et al., 2002) in the Rar1 wild-type background using a modi-fied single cell transient gene expression assay (Azevedo et al., 2002).After delivery of plasmid DNAs into epidermal cells, detached barleyleaves were incubated for 48 h (open bars) or 96 h (closed bars). Subse-quently, leaves were inoculated with spores of Bgh isolate A6 (AvrMla6)and incubated for another 48 h. Microscopic scoring of single interac-tion sites was identical to that described for Figure 2. Asterisks indicatehaustorium indices that are significantly different (P � 0.05) from bom-bardments using empty dsRNAi vector controls. GUS, �-glucuronidase;n.d., not determined.


which may be cued by fungal nitrogen starvation (Van Kan etal., 1991; Perez-Garcia et al., 2001). Here, we have shown thatslow Mla12-triggered resistance was altered dramatically to arapid response by Mla12 overexpression, leading to almostcomplete abortion of Bgh attack before haustorium differentia-tion (Figure 2). Because the rapid response retained AVRMLA12dependence, the Bgh effector protein must be, like AVRMLA1and AVRMLA6 (Halterman et al., 2001; Zhou et al., 2001) (Figure2), delivered before or during the switch from surface to invasivefungal growth. The rapid Mla12 overexpression response sug-gests that cellular amounts of MLA12 or protein complexes con-taining MLA12 are rate limiting for the onset or speed of theresistance. This finding is consistent with previous results dem-onstrating markedly reduced resistance in plants that are het-erozygous for Mla12 (Torp and Jørgensen, 1986). In addition, theretained Rar1 dependence of the Mla12 overexpression pheno-type corroborates this as an authentic response. Assuming thatexpression levels of different Mla genes are similar and sustaincomparable protein abundance, it remains possible that thegene-specific infection types reflect differences in the activitiesof presumed MLA-containing recognition complexes or differentintrinsic activities of AVRMLA proteins.

Determinants of MLA Recognition Specificity

Functional analysis of reciprocal domain-swap constructs be-tween Mla1 and Mla6 revealed an essential role of the LRR-CTunit in specificity determination (Figure 4B). We found that dis-tinct regions in the LRRs of MLA1 and MLA6 (LRRs 3 to 11 and9 to 11, respectively) were necessary for cognate AVRMLA per-ception. This finding is in agreement with LRRs representingthe most variable part of MLA and other characterized NB-LRR–type R proteins (Botella et al., 1998; McDowell et al., 1998; Meyers

et al., 1998; Ellis et al., 1999; Halterman et al., 2001). It also isconsistent with the finding that potentially solvent-exposed res-idues in MLA LRRs and those of other NB-LRR R proteins aresubject to diversifying selection (Botella et al., 1998; McDowellet al., 1998; Meyers et al., 1998; Halterman et al., 2001). One in-terpretation of these data is that the diversified regions are in-volved in ligand-specific recognition.

LRRs have been demonstrated to function as specificity de-terminants of membrane-anchored R proteins (Van der Hoornet al., 2001; Wulff et al., 2001). Successful domain-swap exper-iments have been reported only for intracellular TIR-NB-LRR–encoding resistance alleles at the L locus in flax to the flax rustfungus (Ellis et al., 1999; Luck et al., 2000). Both MLA and Lproteins exhibit comparable average polymorphisms in corre-sponding domains (based on four MLA variants, includingMLA13 [Halterman et al., 2003], and 11 L variants from flax).Unlike our study involving CC-NB-LRR proteins, the analysis ofL chimera functions suggested that both TIR-NB and LRR re-gions can determine specificity differences (Ellis et al., 1999;Luck et al., 2000). Although it is possible that the CC-NB do-main is irrelevant for specificity determination, more divergedCC-NB domains from other MLA proteins must be tested be-fore we can generalize from the observations based on MLA1and MLA6 chimeras.

Reciprocal swaps of the CT domains between MLA1 andMLA6 resulted in nonfunctional chimeras (11116 and 66661;Figure 4B). Our interpretation that cognate LRR-CT units arerequired for MLA specificity determination was supported bythe finding that two of three single–amino acid replacements inmutant MLA12 variants affect CT amino acids and the third af-fects an LRR residue (Figure 1). Additional evidence for a role ofthe MLA CT in specificity determination comes from the identi-fication of a hypervariable region in the middle of this domain

Figure 6. Schemes of the Morex Mla Locus and Genomic Regions Containing Identified Mla Resistance Genes.

DNA sequences encompassing the Morex Mla locus (261 kb, in reverse orientation) (Wei et al., 2002) are represented schematically and drawn toscale in the top line (relevant sequences only). Available genomic sequences of Mla1, Mla6, and Mla12 and flanking regions are shown below. Codingsequences of functional Mla R genes and RGHs are boxed and highlighted in black and blue, respectively. A conserved upstream open reading frame(uORF) and a simple [AT]n microsatellite are shared among functional Mla R genes. Green boxes denote retrotransposon sequences: a BARE1 soloLTR in intron 3 of RGH1bcd, HORPIA2 immediately 3� of RGH1bcd, and ALEXANDRA 5� of RGH1bcd. Dark gray areas denote sequences showing�90% identity, and light gray areas denote sequences showing �75% identity. A possible inversion event could explain the altered relative orienta-tions of homologous genes Mla1-2 and RGH1f as indicated. Note that RGH1e/f and RGH3a/b are extremely similar and located within a 40-kb dupli-cated region (Wei et al., 2002). For this reason, the indicated homologies exist between RGH1e and RGH1f and between RGH3a and RGH3b. Arrowsindicate the relative orientations of genes (5� to 3�). Borders of Morex sequences are indicated in kb according to accession AF427791.

740 The Plant Cell

(residues 893 to 945 in MLA1). This hypervariable region showsan increased ratio of nonsynonymous (ka � 15.4) to synony-mous (ks � 9.6) nucleotide substitutions (based on Mla1, Mla6,Mla12, and Mla13 sequences [Halterman et al., 2003]; signifi-cant at P � 0.1%), which is indicative of the operation of diver-sifying selection. This is like the C-terminal non-LRR domain ofP locus genes that encode flax TIR-NB-LRR proteins, whichalso was found to contain a region that is subject to diversifyingselection and might contribute to specificity determination(Dodds et al., 2001).

RAR1/SGT1 May Act Downstream of Presumptive MLA Recognition Complexes

There is strong evidence suggesting a conserved role for RAR1in R gene–mediated resistance to different pathogen classesand in different plant clades (Shirasu et al., 1999a; Liu et al.,2002b; Muskett et al., 2002; Tornero et al., 2002). RAR1 hasbeen implicated in ubiquitin-protein conjugation pathway(s) to-gether with SGT1 (Azevedo et al., 2002; Liu et al., 2002b). Ubiqui-tination targets have not been identified to date, and it remainsunclear whether RAR1/SGT1 acts upstream of, coincident with,or downstream of R protein recognition complexes. The varia-tion in Rar1 requirement for the function of different Mla resis-tance specificities (Jørgensen, 1996) is unique with regard totheir potential allelism and unusual sequence relatedness. De-spite a dramatic shift to a rapid resistance response resultingfrom the overexpression of Mla12, its Rar1 dependence re-mained unaltered (Figure 2). Likewise, the partial Rar1 require-ment for Mla6 function and the Rar1-independent Mla1 activityremained unchanged upon the expression of both R genesfrom either the strong ubiquitin promoter or native 5� flankingregulatory sequences (Figure 4). Thus, RAR1 dependence ap-pears to be conditioned by subtle intrinsic properties of MLAproteins but not by dosage. Consistent with this finding, re-placement of MLA6 domains with the corresponding MLA1parts generated variants conferring AvrMla6-specific immunitythat was either fully dependent on or independent of Rar1 (Figure4C). We were unable to examine this using the reciprocal chi-meras because these were either nonfunctional (66611) or me-diated Rar1-independent resistance activity (61111 and 66111).

A role for RAR1 in the assembly of preformed R protein–con-taining recognition complexes may be inferred from the findingthat a nonchallenged Arabidopsis rar1 mutant line failed toaccumulate the RPM1 CC-NB-LRR protein to Pseudomonassyringae (Tornero et al., 2002). Our study demonstrates thatthe reliance on RAR1 and SGT1 is not absolute for a given Mlarecognition specificity. Successful uncoupling of AvrMla6 rec-ognition from Rar1/Sgt1 dependence implies that RAR1 cannotbe required for processes that occur “upstream” from recogni-tion (e.g., in planta processing or transport of AVRMLA6) (seechimera 11166 in Figures 4C and 5). Also, the uncoupling ex-cludes the possibility that MLA6 “guards” RAR1 or SGT1 inpresumed MLA-containing recognition complexes. It is possi-ble that the MLA6 CC-NB domain and the LRRs exert antago-nistic roles, the former inhibiting and the latter enhancingRAR1-dependent R protein function (cf. constructs 16666,11166, and wild-type MLA6 in Figure 4C). The observed partial

impairment of Mla6 wild-type function in rar1 plants probably isnot the result of MLA6 destabilization, because the activity wastime independent (unchanged at 15 and 96 h after DNA deliv-ery). This result is consistent with the finding that Mla6 overex-pression in the rar1 mutant background did not increase re-sistance (i.e., the amount of functional recognition complexes)(Figure 4C). Thus, it seems possible that RAR1/SGT1 exerts afunction downstream from activated MLA recognition com-plexes in resistance signaling. Therefore, the observed variationin Rar1/Sgt1 reliance on the function of different MLA wild typeor MLA chimeras may be attributable to variation in signal fluxset by intrinsic activities of MLA variants in AVRMLA-activatedrecognition complexes (e.g., by different half-lives of activecomplexes).

Do MLA Chimeras Affect Folding of MLARecognition Complexes?

The SGT1 binding function of plant RAR1 proteins has beenconserved in monocots and dicots (Azevedo et al., 2002; Liu etal., 2002b). Our data obtained from Sgt1-silencing experimentsin cells expressing MLA chimeras that retain AVRMLA6 recog-nition suggest that RAR1/SGT1 functions in MLA6 resistanceare closely linked (Figure 5). For example, the RAR1-indepen-dent function of chimera 11166 retained full activity upon Sgt1silencing; inversely, chimeras showing full RAR1 dependencealso retained SGT1 dependence. In addition, the function ofMla12, which requires Rar1, was compromised significantly inSgt1-silencing experiments (data not shown). Recent experi-ments using Saccharomyces cerevisiae and Schizosaccharo-myces pombe sgt1 mutant strains indicate a potential role ofthe wild-type protein as a co-chaperone or an assembly factorof diverse regulatory multiprotein complexes, including SCF-type E3 ubiquitin ligases, the structurally related CBF3 kinet-ochore complex, and the Cyr1p adenylyl cyclase complex(Kitagawa et al., 1999, Dubacq et al., 2002; Garcia-Ranea et al.,2002; Schadick et al., 2002). In this context, it is notable that ei-ther of two Arabidopsis SGT1 genes was shown to comple-ment S. cerevisiae sgt1 mutant strains (Azevedo et al., 2002). Acentral conserved part in SGT1 proteins likely adopts a foldsimilar to that of the p23 co-chaperone, which is known to in-teract with the heat-shock protein hsp90 chaperone and partic-ipates in the folding of different regulatory proteins (Dubacq etal., 2002; Garcia-Ranea et al., 2002). Therefore, it is possiblethat the observed variation in RAR1/SGT1 dependence for thefunction of different Mla resistance specificities or MLA chime-ras reflects differences in the degree of folding/activation assis-tance needed for presumed MLA-containing recognition com-plexes. In this scenario, the signal flux in downstream signalingpathways might be similar for both RAR1/SGT1-dependent and-independent resistance.

METHODS

Plant and Fungal Material

Sultan 5 is a chromosome-doubled haploid barley (Hordeum vulgare)cultivar containing Mla12. Mla12 mutants (M66 and M86), a rar1-2 mutant


allele (M100), and the rar2 mutant (M22) were generated by chemicalmutagenesis of Sultan 5 seeds (Torp and Jørgensen, 1986). Ingrid (mlo-3Rar1) was generated by seven backcrosses with cv Ingrid, and the mlo-29 rar1-2 double mutant was isolated originally from a remutagenizedrar1-2 M2 population. The latter line was used to test the Rar1 depen-dence of MLA chimeras (Figure 4). All barley seedlings were grown at20�C with 16 h of light and 8 h of darkness. The barley powdery mildew(Blumeria graminis f. sp. hordei [Bgh]) isolate A6 (AvrMla6, AvrMla12,virMla1) was maintained on P01, a nearly isogenic line from cv Pallascontaining Mla1. Isolate K1 (AvrMla1 virMla6 virMla12) was maintainedon I10, a nearly isogenic line from cv Ingrid containing Mla12. Plants ordetached leaves were kept at 18�C and 60% RH with 16 h of light and8 h of darkness after inoculation with Bgh spores.

Genomic Library Construction and Screening forMLA12-Containing Cosmids

High molecular mass genomic DNA was isolated from Sultan 5 contain-ing Mla12 and partially digested with Sau3AI to produce DNA fragmentsof 30 to 60 kb. After dephosphorylation, the fragments were ligated tothe XbaI-BamHI–linearized SuperCos cosmid vector according to themanufacturer’s instructions (Stratagene). A total of 240 pools averaging4000 clones each were made and kept frozen as glycerol stocks. The li-brary had an average insert size of 25 kb (ranging from 15 to 40 kb) andrepresents approximately five genome equivalents. DNA preparationswere made using the R.E.A.L Prep 96 Plasmid Kit (Qiagen, Valencia, CA)from all pools. For library screening, the plasmid DNA of each pool wasdigested with HindIII or EcoRI, resolved by 0.8% agarose gel electrophore-sis, and blotted onto Hybond-N� membranes (Amersham Pharmacia Bio-tech). To identify positive pools containing the Mla12 candidate gene, theDNA gel blots were hybridized with a 32P-labeled probe, which was derivedfrom the Leu-rich repeat region of Mla1 (covering exon 4 of Mla1) (Zhou etal., 2001). Approximately 15,000 colonies of each positive pool werescreened by hybridization with the same probe to obtain purified clones.Positive clones were fingerprinted using various restriction enzymes.

Sequencing and Gene Characterization

Plasmid DNA of Mla12-containing clones was isolated using the Midi-Plasmid-DNA Prep Kit (Qiagen), subcloned, and sequenced as de-scribed (Zhou et al., 2001). Construction of sequence contigs was per-formed using the GCG9 and STADEN software packages (University ofWisconsin Genetics Computer Group, Madison). Sequence alignmentwas performed using a World Wide Web–based program (http://prodes.toulouse.inra.fr/multalin/multalin.html).

Sequencing of Mla12 Mutant Alleles

Genomic DNA was isolated from the Mla12 mutants M86 and M66 andthe rar2 mutant M22. The DNA was used as a template for PCR amplifi-cation of the respective Mla12 mutant alleles. Mla12-specific primerswere designed based on the sequence alignment of Mla12, Mla1, Mla6,Mla1-2, and RGH1a (primer sequences are listed in Table 1). PCR prod-ucts were purified using the Qiagen PCR Product Purification Kit andthen sequenced directly. Mutations were identified by aligning the se-quences of PCR products to Mla12 and confirmed by three additional in-dependent PCR procedures and sequencing of plus and minus strands ofthe mutated region.

Construction of Mla-Containing Plasmid Expression Vectors

pUbi-GFP-Nos [maize ubiquitin1 promoter-GFP-Nos poly(A) signal](Shirasu et al., 1999b) was used as a backbone to subclone Mla1, Mla6,

and Mla12. The green fluorescent protein open reading frame was de-leted using restriction enzymes PstI and SacI and replaced by an adap-tor with a suitable multiple cloning site for Mla genes. The 5� untranslatedregion of Mla1 was amplified by PCR using primer pairs MlapstIs1 andMlaAgeIas1, and the product was cloned into pGEM-T vector (Promega)and confirmed by sequencing. The 5� untranslated regions were sub-cloned into the pUbi-Adaptor-Nos vector using enzymes HindIII andAgeI. The 3� untranslated region of Mla1 was amplified with primersMlaEcoRIas1 and MlaBsrDIs1 and cloned into pGEM-T vector. Aftersequence confirmation, the 3� untranslated region was subcloned intothe pUbi-Adaptor-Nos vector using BsrDI and NotI. The plasmid vectorthen was linearized with AgeI and BsrDI, and coding regions of Mla1,Mla6, and Mla12, including introns 3 and 4, were inserted. The resultingoverexpression plasmids were designated pUbiMla1Nos, pUbiMla6Nos,and pUbiMla12Nos. They served as backbones to generate domain-swap constructs between Mla1 and Mla6 and Mla mutant variant con-structs (see below). Plasmids driven by native 5� flanking Mla pro-moter sequences were generated by subcloning an 8-kb SacII-XhoIfragment from Mla1 containing cosmid p6-49-15, or an AvrII-PciIfragment of Mla6 containing cosmid 9589-5a, into pBluescript II KS

(Stratagene).Plasmids 16666 and 61111 were generated by exchanging BbsI-NotI

fragments, which were derived from pUbiHEMla1Nos and pUbiHEMla6Nos,respectively. Likewise, plasmids 11666 and 66111 were generated byexchanging Bsu36I-NotI fragments. Plasmids 11166 and 66611 weregenerated by splicing by overlap extension (SOE) using the forwardprimer MlaBbSIs, the reverse primer NotIas, and the overlapping primersP10s and P10as. The Bsu36I-NotI enzyme pair was used to digest theSOE products that were inserted into pUbiMla1Nos or pUbiMla6Nos di-gested with the same enzyme pair, respectively. Plasmids 11661 and66116 also were generated by SOE with primers P5s/P5as and P12s/P12as covering the swap sites and the flanking primers MlaBbSIs andMla1EcoRIas1. The BbsI-NotI–digested fragments of the SOE productswere inserted into pUbiMla1Nos and pUbiMla6Nos, respectively. Plas-mids 11116 and 66661 were generated by subcloning Bsu36I-NotIfragments of plasmids 66116 and 11661 into pUbiMla1Nos andpUbiMla6Nos, respectively.

Table 1. Mla12-Specific Primer Sequences

Primer Sequence Positionin Mla12

Mla12S1a 5�-CACCTCACCTTCTGTCTCTCTC-3� 488Mla12S1b 5�-GCATCTTTCTTGCTATTCTGCTC-3� 328Mla12S1c 5�-TGCCATTTCCAACCTGATTCCC-3� 12Mla12AS1a 5�-CCTTGTTCCTGTCACGCCTATC-3� 34Mla12AS1b 5�-CCTTTAATCTTCTCGTATACCGCTC-3� 658Mla12AS1c 5�-TGTTTAGTGTGAACTGCTTATGCC-3� 945Mla12As1d 5�-TCTCCCTCTTTCCTTCCTCTCC-3� 1228Mla12S2a 5�-GATGCTTAATGAGAGTAAGATTATCGAG-3� 1705Mla12S2b 5�-GGCATCAACTTTGCTTTCTCCAATAG-3� 1913Mla12AS2b 5�-CGACGACAATTACTCTGTGAAGAC-3� 2652Mla12AS2a 5�-GAAGGGACAAACGACGACAATTACT-3� 2663Mla12S3a 5�-TAACAGTTTAGAGGAGATGCGG-3� 2366Mla12S3b 5�-CTCCCGACTGAGATAGGAAAAC-3� 2915Mla12S3c 5�-TTGTTGTCCCTTCGTCGTCTCTGG-3� 3586Mla12AS3b 5�-CACAATAGAGAAGAACAAAGACATC-3� 3775Mla12AS3c 5�-TGTGCGCCAAAAATCAGTTCTCAC-3� 4057Mla12AS3a 5�-ATGGAGAAAGGAAGGTAGGTGG-3� 4139

742 The Plant Cell

For the construction of plasmids pUbiMla1(K915M), pUbiMla6(K913M), and pUbiMla12(K916M), a single amino acid exchange wasintroduced by SOE using a template of pUbiMla1Nos, pUbiMla6Nos,and pUbiMla12Nos, respectively. Likewise, variants pUbiMla1(L631R),pUbiMla6(L631R), and pUbiMla12(L631R) were generated by SOE reac-tions using the same template DNAs. Primers used for these reactionsare listed in Table 2. For site-directed mutagenesis of the codon leadingto the replacement of Lys with Met, two common primers, MLABbSIsand MLABsrDIas1 (for Mla1 and Mla6) and MLA12BsrDIas1 (for Mla12),were used in combination with overlapping primers MLA12DNas2 andMla12DNs1. The BbSI-BsrDI enzyme pair was used to digest the SOEproducts, and the resulting fragments were inserted into pUbiMla1Nos,pUbiMla6Nos, and pUbiMla12Nos. For site-directed mutagenesis of thecodon leading to the replacement of Leu with Arg, four common primers,P2s, M66-as, M66-s, and Exon-5as, were used for SOE reactions. TheSOE products were digested with Bsu36I-SbfI (for Mla1 and Mla12) orBsu36I-BspEI (for Mla6), and fragments were inserted into pUbiMla1Nos, pUbiMla6Nos, and pUbiMla12Nos digested with the same enzymepair, respectively.

Single-Cell Transient Expression Assay

The single-cell transient expression assay was performed essentially ac-cording to Shirasu et al. (1999b). Reporter plasmids containing Mlo and�-glucuronidase (GUS) genes (GUS alone in the case of the Mlo geneticbackground) and the respective effector plasmids were mixed beforecoating of the particles (molar ratio of 2:1; 5 g of total DNA). The bom-barded leaves were transferred to 1% agar plates supplemented with 85M benzimidazole and incubated at 18�C for 15 h before high-density in-oculation with Bgh spores. Leaves were stained for GUS, and single leafepidermal cells attacked by Bgh germlings were evaluated microscopi-cally at 48 h after spore inoculation. In the double-stranded RNA interfer-

ence single-cell silencing experiments, particles were co-coated with aconstruct encoding an intron-spliced double-stranded RNA interferenceconstruct targeting HvRAR1 or HvSGT1 according to Azevedo et al.(2002) (molar ratio of 1:1:1; 5 g of total DNA). Note that in the gene-silencing experiments, the bombarded leaves were inoculated at 18�Cfor 48 or 96 h before high-density inoculation to allow the turnover ofpreformed RAR1 or SGT1.

Upon request, all novel materials described in this article will be madeavailable in a timely manner for noncommercial research purposes.

Accession Number

The GenBank accession number for the Mla12 genomic sequence isAY196347.

ACKNOWLEDGMENTS

We thank Roger Wise and Dennis Halterman for the gift of C.I. 16151cosmid clone 9589-5a containing Mla6 and for sharing associated se-quence data before publication. We thank Jane Parker for providing valu-able comments on the manuscript. This work was supported by the MaxPlanck Society and the Gatsby Charitable Organization.

Received November 14, 2002; accepted December 29, 2002.

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Table 2. Primers Used in SOE Reactions

Primer Sequence

Exon-5as 5�-AATCGTCATCATGAGCACCTT-3�

M66-as 5�-CCAACACCTCCAAAAACTGCCGTTTTCCT-3�

M66-s 5�-CTGAGATAGGAAAACGGCAGTTT-3�

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Mla12BsrDIs1 5�-ACATTGCATCAGATGTGCTCTG-3�

Mla12DNas2 5�-GCTTCCATTGCCTCCCCAACCCT-3�

Mla12DNs1 5�-GAGCGAGGGTTGGGGAGGCAATG-3�

Mla1EcoRIas1 5�-AAGCGGCCGCGAATTCTAATACTACTAGGA-CTCG-3�

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MlaBbSIs 5�-TGGGAATAGCATGTCTTCACAG-3�

MlaBsrDIas1 5�-TGATGCAATGTGAGTCGCTCTGG-3�

MlaBsrDIs1 5�-CTGATCCAGAGCGACTCACATTGC-3�

MlaPstIs1 5�-CTTCTGCAGACTGAGTCATCGGCACCTTGC-3�

NotIas 5�-GCAAGACCGGCAACAGGATTCAA-3�

P10as 5�-TCGCAGTGCAGAGAGTTGGCT-3�

P10s 5�-AGCCAACTCTCTGCACTGCGA-3�

P12as 5�-TCAAACAATATCTGCGTGGCA-3�

P12s 5�-TGCCACGCAGATATTGTTTGA-3�

P2s 5�-GCTCGGATTAAATTACTTCAACC-3�

P5as 5�-CAAGATCCAACACCTCCAAAAACT-3�

P5s 5�-AGTTTTTGGAGGTGTTGGATCTT-3�


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148

11. Acknowledgements

I find I owe a debt to many people when I reflect on all the way of

pursuing my Ph.D study. However, it is more appropriate to acknowledge

those I know have directly or indirectly contributed to this work.

There is one person who turned the track of my ‘academic life’ -

although it is still too early to use these words - by allowing me working on

MLA and the way of his superb guidance. My sincere gratitude goes to Prof.

Dr. Paul Schulze-Lefert, my supervisor, for his excellent advices, unlimited

encouragement and support throughout my study. It was difficult to know what

is ‘Consistency and Precision’ for scientific work, however, it will become

easier to apply them in my future career.

There are some other people whose help was so precious. I

particularly thank Dr. Staphane Bieri for his valuable support when I moved to

Köln and his discussions that proved to be fruitful. I would like also to thank

Dr. Fasong Zhou for his help when I started my doctorate training in

Sainsbury Laboratory, John Innes Centre, England in 2001. I am also grateful

to Dr. Ken Shirasu for hosting me in Sainsbury Lab and for extending his

generous assistance upto the present time. I would like to thank other MLA

members, Stefan Mauch, Francesca Ceron and Simone Pajonk. Thanks for

the nice time working together and helping each other. I have been also

benefited a lot from other colleagues, Drs. Stephan Bau, Riyaz Bhat, Judith

Müller and Ralph Panstruga, as well as Chiara Consonni, Jan Dittgen,

Häweker Heidrun, Marco Mikilis, and Anja Reinstädler. They are so helpful in

the lab or during the preparation of this thesis. I also appreciate the help from

other colleagues in MPIZ, special thanks to Drs. Laurant Deslandes and

Joachim Uhrig for helped me on yeast work.

I realize that it would have been impossible to take care of both my

work and myself without the unbelievable support from my wife, for this she

deserves all credits that I deserve for the completion of the study.

149

"Ich versichere, daß ich die von mir vorgelegte Dissertation selbständig

angefertigt, die benutzten Quellen und Hilfsmittel vollständig angegeben und

die Stellen der Arbeit - einschließlich Tabellen, Karten und Abbildungen -, die

anderen Werken im Wortlaut oder dem Sinn nach entnommen sind, in jedem

Einzelfall als Entlehnung kenntlich gemacht habe; daß diese Dissertation

noch keiner anderen Fakultät oder Universität zur Prüfung vorgelegen hat;

daß sie - abgesehen von unten angegebenen Teilpublikationen - noch nicht

veröffentlicht worden ist sowie, daß ich eine solche Veröffentlichung vor

Abschluß des Promotionsverfahrens nicht vornehmen werde. Die

Bestimmungen dieser Promotionsordnung sind mir bekannt. Die von mir

vorgelegte Dissertation ist von Prof. Dr. Paul Schulze-Lefert betreut worden."

Köln, im May 2004

Ein Teil dieser Arbeit wurde bereits veröffentlicht:

Shen, Q.-H., Zhou, F.S., Bieri, S., Haizel, T., Shirasu, K., and Schulze-Lefert, P. (2003). Recognition specificity and RAR1/SGT1

dependence in barley Mla disease resistance genes to the powdery

mildew fungus. Plant Cell 15, 732-744.

Ein Teil dieser Arbeit wird zur Veröffentlichung vorbereitet:

Bieri, S., Shen, Q.-H., Mauch, S., and Schulze-Lefert, P. Barley MLA

protein abundance is controlled by RAR1 and is rate-limiting for effieient

resistance to the powdery mildew fungus. Manuscript in preparation.

150

12. Lebenslauf

ADDRESSE: Kolibriweg 14, Köln,D-50829 NATIONALITÄT: Chinesisch Tel.: +49(0)221 5062 329

GEBURTSTAG: 15. Oktober 1964, männlich FAMILIENSTAND: Verheiratet

AUSBILDUNG

Seit 2001 MAX PLANCK INSTITUT FÜR ZÜCHTUNGSFORSCHUNG (MPIZ)

Abteilung Molekulare Phytopathologie, Köln, Deutschland

& THE SAINSBURY LABORLATORY, Norwich, England

1999 – 2000 JOHN INNES CENTRE & UNIVERSITY OF EAST ANGLIA

School of Biological Sciences, Norwich, England

M.Sc.: Pflanzenzüchtung und Biotechnologie

Projekt: Lokalisierung von Nichtwirtsresistenzgenen gegen

Gelbrost in Weizen mittels QTL-Analyse

1985 – 1987 HUAZHANG UNIVERSITÄT FÜR AGRARWISSENSCHAFTEN Abteilung für Agrarwissenschaften, Wuhan, China

M.Sc.: Pflanzengenetik und -züchtung

Projekt: Quantitative genetische Analyse von

Ertragsmerkmalen in Gerste

1981 – 1985 JIANGXI UNIVERSITÄT FÜR AGRARWISSENSCHAFTEN

Abteilung für Agrarwissenschaften, Nanchang, China.

Bachelor of Sciences für Agrarwissenschaften

BERUFSERFAHRUNG

1998 – 1999 GASTWISSENSCHAFTLER Abteilung für Biotechnologie, University of Westminster,

London

1993 – 1998 DOZENT School of Biological Sciences, Nanchang Universität, China

1992 – 1993 ASSISTENT DES DIREKTORS QC/QA, Nanchang Biotech Company, China

1987 – 1992 HILFSDOZENT School of Biological Sciences, Jiangxi Universität, China

Functional Analysis of Barley MLA-triggered Disease ... · 2.2.4. Protein analysis 2.2.4.1....

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